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164 publications mentioning mmu-mir-199a-2 (showing top 100)

Open access articles that are associated with the species Mus musculus and mention the gene name mir-199a-2. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1
[+] score: 475
Comparison of the gene expression profiles obtained in lung fibroblasts transfected with miR-199a-5p precursors or with a siRNA specifically directed against CAV1 revealed an overlap between the 2 signatures, mainly among the down-regulated transcripts (Figure S10A, group 2): 34% of miR-199a-5p downregulated transcripts were also repressed by a siCAV1 (Figure S10B). [score:10]
Finally, in contrast to a recent report showing that miR-199a-5p, by targeting SMAD4, inhibited TGFβ -induced gastric cancer cell growth [53], we found that lung fibroblasts overexpressing miR-199a-5p have an increased SMAD4 expression (Figure S14B), suggesting thus a potential opposite function of this miRNA between epithelial and mesenchymal cells. [score:9]
Taken together, these experiments show that the observed up-regulation of miR-199a-5p expression in the fibrotic lungs of mice is correlated with a downregulation of CAV1. [score:9]
Of note, BALB/c mice, for which pulmonary expression of miR-199a-5p was not upregulated in response to bleomycin, did not display a significant decrease in CAV1 mRNA expression level 14 days after bleomycin treatment (Figure S7). [score:8]
List of the main miR-199a-5p predicted targets significantly downregulated following miR-199-5p overexpression in human fibroblasts using the bioinformatics tool miRonTop (http://www. [score:8]
Among the set of transcripts that were down-regulated after ectopic expression of miR-199a-5p, we then restricted our work to a group of 21 miR-199a-5p target genes predicted by 3 independent algorithms, showing the largest modulation factors and smallest statistical p-values. [score:8]
A specific overrepresentation of predicted targets for miR-199a-5p and miR-21 in the population of down-regulated transcripts was noticed after heterologous expression of either miR-199a-5p or miR-21, respectively. [score:8]
Normal human pulmonary fibroblasts MRC5 were transfected with a control LNA inhibitor (LNA-CT), a LNA-miR-199a-5p inhibitor or a target site blocker directed against CAV1 3-UTR (CAV1 protector) (n = 2). [score:8]
Among these miRNAs, 3 were downregulated (miR-193, miR-30b and miR-29c) and 2 were upregulated (miR-199a-3p and miR-199a-5p) (Figure 9B). [score:7]
Given that loss of CAV1 expression represents a critical factor involved in the fibrogenic activation of pulmonary fibroblasts [19], we assessed whether overexpression of miR-199a-5p in lung fibroblasts was sufficient to recapitulate known profibrotic effects associated with a decrease in CAV1 expression (i. e. ECM synthesis, fibroblasts proliferation, migration, invasion and differentiation into myofibroblasts) [30], [32], [33]. [score:7]
Of note, these two downregulated genes were predicted to be direct targets of miR-199a-5p by Pictar. [score:7]
We then focussed our analysis on a subset of 21 transcripts containing miR-199a-5p complementary hexamers in their 3′UTR, showing the largest inhibition of expression, and identified by TargetScan, PicTar and miRanda (Figure 2C and Table 1). [score:7]
Microarray analysis of lung fibroblasts transfected with 10 nM of miR-199a-5p mimic or miR-Neg reveals a significant increase of ACTA2 expression (*p<0.05), a hallmark of myofibroblast differentiation, as well as a significant decrease of PPARG expression (**p<0.01), a known inhibitor of myofibroblast differentiation. [score:7]
Thus, altered expression of miR-199a-5p is likely to represent a primary pathogenic mechanism in the development of lung fibrosis rather than a secondary effect of the long-standing disease process. [score:6]
We thus anticipate that strategies preventing the up-regulation of miR-199a-5p may represent a new effective therapeutic option to treat fibroproliferative diseases. [score:6]
As depicted in Figure 4D and S6, both LNA -mediated silencing of miR-199a-5p and blocking miR-199a-5p binding on CAV1 3′UTR inhibit TGFβ -induced downregulation of CAV1. [score:6]
Interestingly, these regulations were independent of CAV1 targeting, suggesting therefore that miR-199a-5p modulates the expression of several components of various distinct biological pathways to elicit, in lung fibroblasts, a profibrotic response. [score:6]
In particular, compared to siCAV1 transfected cells, overexpression of miR-199a-5p significantly increased CCL2, TGFBRI and MMP3 expression and significantly decreased CAV2 and PLAU expression (Figure S12). [score:6]
As TGFβ is known to downregulate CAV1 in pulmonary fibroblasts [19], we then investigated whether decreased expression of CAV1 upon TGFβ stimulation was associated with an increase in miR-199a-5p expression. [score:6]
To further investigate whether miR-199a-5p is involved in TGFβ -induced downregulation of CAV1, we performed additional experiments using a LNA -based inhibitor of miR-199a-5p as well as a CAV1 target site blocker to specifically interfere with miR-199a-5p binding on CAV1 3′UTR. [score:6]
Moreover, this inhibition was also repeated using the whole 3′-UTR of human CAV1 (Figure S4), demonstrating that CAV1 is indeed a direct target of miR-199a-5p. [score:6]
Based on previous studies that demonstrated a significant link between the downregulation of caveolin-1 (CAV1) in lung fibroblasts and the deleterious effects mediated by TGFβ [19], [30], CAV1 represented a particularly relevant putative miR-199a-5p target gene. [score:6]
As detected by Taqman RT-PCR, TGFβ treatment of human fibroblasts for 24 h or 48 h caused a marked decrease of CAV1 mRNA, whereas miR-199a-5p expression was significantly upregulated (Figure 4A and 4B). [score:6]
Furthermore, we showed that TGFβ exposure of stellate cells was associated with an increase of miR-199a-5p expression and a decrease of CAV1 expression level (Figure 10E and 10F). [score:5]
While both miR-21 and miR-199a-5p have been shown to be regulated by TGFβ, their expression may be primarily regulated through a Smad -dependent post-transcriptional mechanism promoting miRNA maturation by Drosha [59], [60]. [score:5]
The mean normalized fluorescence intensity for the agilent probe is displayed; (B) Western blot analysis showing the effect of miR-199-5p or miR-199a-3p overexpression in hFL1 lung fibroblasts on SMAD4 and αSMA expression. [score:5]
For miR-199a-5p knockdown and target site blocker experiments, anti-miR-199a-5p LNA, negative control anti-miR-159s LNA (miRCURY LNA Knockdown probes) and CAV1 protector were ordered from Exiqon. [score:5]
Finally, heterologous expression of miR-199a-5p also led to a strong increase in α smooth mucle actin (αSMA) expression (Figure 7E and Figure S9), a hallmark of myofibroblast differentiation as well as to a significant potentiation of COL1A1 induction in response to TGFβ (Figure 7F). [score:5]
Finally, examination of IPF lung sections revealed a specific expression of miR-199a-5p in fibroblastic foci of the injured lung as well as a decreased CAV1 expression (Figure 6C and 6D). [score:5]
Figure S9Expression of ACTA2 and PPARG following overexpression of miR-199a-5p in lung fibroblasts. [score:5]
Figure S14 Expression of miR-199a-3p in lung fibrosis and impact of its overexpression on pulmonary fibroblast differentiation. [score:5]
Interestingly, as for lung fibrosis, kidney expression of miR-199a-5p was correlated with disease progression. [score:5]
In a recent report describing the miRNA expression profile of lung fibroblasts, miR-199a-5p was found to be highly expressed [25]. [score:5]
Our systematic analysis of the gene expression profiles of lung fibroblasts overexpressing miR-199a-5p led to the identification of a large number of transcripts that were significantly modulated by this miRNA. [score:5]
Expression of miR-199a-5p expression was increased in lungs of IPF patients (GEO accession number GSE13316 from [13]; dataset consisting of ten IPF samples and ten control samples; two different probes for miR-199a-5p with a p-value of p = 0.005 and p = 0.006, wilcoxon rank sum test, Table S3). [score:5]
While our data also showed an enhanced pulmonary expression of these two miRNAs in the bleomycin -induced mouse mo del, expression of miR-199a-5p was more significant in IPF samples (Table S3 and Figure S14A). [score:5]
Additionally, we showed in a large cohort of IPF patients an enhanced expression of miR-199a-5p that was reproduced in three independent mouse mo dels of fibrosis as well as a decreased expression of CAV1. [score:5]
Altogether, these experiments demonstrate that, in lung fibroblasts, induction of miR-199a-5p in response to TGFβ mediates CAV1 downregulation through binding on a unique site located in CAV1 3′UTR. [score:4]
To investigate the regulatory mechanisms underlying miR-199a-5p production, we assessed the expression status of the 2 mouse genes, miR-199a-1 (on chromosome 9) and miR-199a-2 (on chromosome 1) in response to bleomycin using a Taqman assay designed to discriminate between pri-miR-199a-1 and pri-miR-199a-2. Our results showed that, 14 days after bleomycin instillation, both pri-miR-199a transcripts were up-regulated in the lungs of C57BL/6 mice (Figure S2B) and thus, contributed to miR-199a-5p production. [score:4]
To address this question, we first attempted to identify gene targets and cellular pathways regulated by miR-199a-5p using the methodology described earlier [25], [26]. [score:4]
Importantly, several profibrotic genes were specifically regulated by miR-199a-5p and their altered expression was confirmed in vivo (Figure S11 and Table S4). [score:4]
Figure S11Comparison of gene expression changes between miR-199a-5p-regulated genes in hFL1 human lungs fibroblasts and lungs from C57BL/6 mice 14 days after bleomycin injection. [score:4]
Overall we showed that miR-199a-5p is a major regulator of fibrosis with strong therapeutic potency to treat fibroproliferative diseases such as IPF. [score:4]
miR-199a-5p and its target CAV1 are dysregulated in IPF. [score:4]
CAV1 is a direct target of miR-199a-5p. [score:4]
TGFβ regulates CAV1 by increasing miR-199a-5p expression. [score:4]
Among these miRNAs, miR-199a-5p was found to be selectively up-regulated in myofibroblasts of the injured lung in bleomycin -treated mice and fibroblastic foci of IPF patients. [score:4]
Production of miR-199a-5p in response to TGFβ in lung fibroblasts results in CAV1 downregulation and subsequently, impaired TGFβ/TGFβR complex degradation. [score:4]
In lung fibroblasts, miR-199a-5p acts as an effector of TGFβ signaling, regulates CAV1 expression, a critical mediator of the lung fibrosis process [18]– [21] and participates to multiple fibrogenic associated-processes including cell proliferation, migration, invasion and differentiation into myofibroblasts. [score:4]
This analysis revealed a significant overlap between these two signatures, as assessed by normalized enrichment scores above 1 (1.4 and 2.17 for up- and down-regulated genes respectively, with nominal p-value and FDR q-value being <0.05), suggesting therefore, that miR-199a-5p is involved in the TGFβ response of lung fibroblasts (Figure 8B). [score:4]
miR-199a-5p mediates TGFβ -induced downregulation of CAV1 in pulmonary fibroblasts. [score:4]
These experiments have established that miR-199a-5p is directly or indirectly involved in the regulation of genes previously associated with lung fibrosis: CCL2, a potent mononuclear cell chemoattractant, PLAU [54], a component of the fibrinolysis system, TGFBRI, the TGFβ receptor type I [55], MMP3 [56] and CAV2 [57]. [score:4]
Invasion of MRC5 fibroblast overexpressing miR-199a-5p was assessed using commercially available 24-well BioCoat Matrigel Invasion Chamber (BD Biosciences). [score:3]
Figure S2 miR-199a-5p and pri-miR-199a expression in C57BL/6 mice 14 days following bleomycin exposure. [score:3]
Increased expression of miR-199a-5p in lung fibroblasts results in an enhanced ability of fibroblasts to migrate, invade matrigel, proliferate and differentiate into myofibroblasts. [score:3]
Altered expression of miR-199a-5p in CCl [4] induced liver fibrosis mouse mo del. [score:3]
Liver fibrosis was induced by injection of CCl [4] and expression of miR-199a-5p was assessed 6 weeks after CCl [4] treatment as well as 2 and 4 weeks after the last injection. [score:3]
Forty-eight hours after ectopic overexpression of each miRNA, a significant alteration (defined by an absolute log [2]ratio above 0.7 and an adjusted p-value below 0.05) of 1261 and 753 transcripts was detected in the miR-199a-5p and miR-21 conditions, respectively. [score:3]
The enhanced expression of miR-199a-5p was confirmed in two independent experimental mo dels of liver fibrosis (Figure 10A–10C) and was correlated with the severity of liver fibrosis, as BALB/C mice have a more pronounced liver fibrosis than C57BL/6 mice, following administration of CCL [4] (Figure 10A and 10B). [score:3]
The recently reported association of CAV1 with kidney fibrosis [61], [62], together with the exclusive distributions of miR-199a-5p and CAV1 in the injured kidney, leads us to hypothesize that miR-199a-5p also controls CAV1 expression in kidney, thus contributing to kidney fibrosis. [score:3]
miR-199a-5p expression during bleomycin induced lung fibrosis. [score:3]
Figure S13Enhanced expression of miR-199a-5p in clinical samples from patients with liver fibrosis. [score:3]
Other miRNAs candidates including miR-214, clustered with miR-199a-2 on mouse chromosome 1 as well as other miRNAs that have been previously associated to fibrosis, including miR-221-222 and miR-449a [37], [38] also showed an enhanced expression in the lung fibrosis-susceptible mice. [score:3]
Concomitant altered expression of both CAV1 and miR-199a-5p in lungs of IPF patients. [score:3]
We exposed the MRC-5 cell line to TGFβ, and analyzed the expression levels of CAV1 and miR-199a-5p. [score:3]
1003291.g003 Figure 3 (A) Position of miR-199a-5p target site in CAV1 3′ UTR and sequence alignment of miR-199a-5p and the CAV1 3′ UTR from various species are shown. [score:3]
1003291.g007 Figure 7Increased expression of miR-199a-5p in lung fibroblasts results in an enhanced ability of fibroblasts to migrate, invade matrigel, proliferate and differentiate into myofibroblasts. [score:3]
1003291.g006 Figure 6(A,B) Box plots showing the normalized expression of log [2]-transformed CAV1 and miR-199a-5p in both IPF (n = 94) and control (n = 83) lungs. [score:3]
In addition, cell cycle analysis (percent cells in S phase) showed that proliferation rate of pulmonary fibroblasts overexpressing miR-199a-5p was significantly enhanced (Figure 7D). [score:3]
Table S1 List of themes corresponding to “canonical pathways” annotations identified by Ingenuity Pathway Analysis in response to overexpression of miR-199a-5p or miR-21 in human pulmonary fibroblasts hFL1. [score:3]
Table S3 Pulmonary expression of miR-199a-5p in 10 IPF and 10 controls (dataset GEO accession number GSE13316). [score:3]
In addition, in situ hybridization experiments performed in the injured lungs from C57BL/6 mice 14 days after bleomycin instillation revealed a selective expression of miR-199a-5p in myofibroblasts (Figure 1C). [score:3]
Interestingly, enhanced expression of miR-199a-5p was also observed in clinical samples from patients with liver fibrosis (Figure S13). [score:3]
1003291.g010 Figure 10Altered expression of miR-199a-5p in CCl [4] induced liver fibrosis mouse mo del. [score:3]
This is further demonstrated by overexpression of miR-21 and miR-199a-5p, which induce lung fibroblast migration to a similar extent (Figure S15). [score:3]
Real Time TaqMan PCR showing the levels of (A) mature miR-199a-5p and miR-199a-3p; (B) pri-miR-199a-1 and pri-miR-199a-2. n = 2. Data are expressed as mean ± SEM *p<0.05. [score:3]
Figure S5 Decreased CAV1 expression after transfection of MRC5 lung fibroblasts with pre-miR-199a-5p. [score:3]
Identification of miR-199a-5p target genes in lung fibroblasts. [score:3]
To further demonstrate the importance of miR-199a-5p in TGFβ response, silencing of miR-199a-5p was performed in lung fibroblasts using LNA -based inhibitors. [score:3]
Identification of miR-199a-5p candidate targets using a transcriptomic approach. [score:3]
CAV1 is a bona fide miR-199a-5p target. [score:3]
The influence of miR-199a-5p on human pulmonary hFL1 fibroblast transcriptome was compared with that of miR-21, which has been previously associated with the development of fibrotic diseases including lung fibrosis [14], [15], [27] (Data set 2, GEO accession number GSE34815). [score:3]
Altered expression of miR-199a-5p and CAV1 in the unilateral ureteral obstruction (UUO) mouse mo del of kidney fibrosis. [score:3]
1003291.g011 Figure 11(A) miR-199a-5p expression in kidney from C57BL/6 mice after UUO at the indicated time points. [score:3]
CAV1 is a bona fide miR-199a-5p targetAlignment of miR-199a-5p with human CAV1 3′UTR sequence revealed one potential conserved seed site (Figure 3A). [score:3]
In particular, we showed that LNA -mediated silencing of miR-199a-5p strongly inhibited TGFβ -induced differentiation of lung fibroblasts into myofibroblasts (Figure 8C and Figure S6), SMAD signaling (Figure 8D) and stimulation of wound repair (Figure 8E and 8F). [score:3]
Figure S12Profibrotic genes significantly modulated in lung fibroblasts by miR-199a-5p independently of CAV1 regulation. [score:2]
MRC-5 lung fibroblasts were treated with 10 ng/mL TGFβ for 24 h and 48 h. MiR-199a-5p (A) and CAV1 expression levels (B) were determined by Taqman PCR. [score:2]
We next sought to determine the mechanism by which miR-199a-5p dysregulation may lead to tissue fibrosis. [score:2]
Figure S8 MiR-199a-5p expression determined by qPCR using FFPE lung samples. [score:2]
1003291.g004 Figure 4 MRC-5 lung fibroblasts were treated with 10 ng/mL TGFβ for 24 h and 48 h. MiR-199a-5p (A) and CAV1 expression levels (B) were determined by Taqman PCR. [score:2]
Table S4 List of the 133 genes modulated by miR-199a-5p in lung fibroblasts that are also dysregulated in lungs from C57BL/6 mice 14 days after bleomycin treatment. [score:2]
Finally, our observation that miR-199a-5p is also dysregulated in two additional experimental mo dels of tissue fibrosis (i. e. kidney fibrosis and liver fibrosis) establishes miR-199a-5p as a ubiquitous factor associated with tissue fibrogenesis. [score:2]
Box plot showing the increased expression of miR-199a-5p in IPF samples (n = 10) compared to control (n = 10). [score:2]
This analysis revealed some proximity between miR-199a-5p and siCAV1 based on the existence of shared regulated pathways (Figure 8A). [score:2]
In line with this, we provide evidence that miR-199a-5p can directly repress CAV1 in lung fibroblasts, thereby stimulating their proliferation, migration, invasion and differentiation into myofibroblasts (Figure 12). [score:2]
Remarkably, similar results were obtained using CAV1 protector, demonstrating therefore that miR-199a-5p is a key effector of TGFβ response through CAV1 regulation (Figure 8C, 8D, 8E, 8F and Figure S6). [score:2]
The probability to obtain the number of genes in a certain pathway in the list of differentially expressed genes between either miR-199a-5p or miR-21 versus miR-Neg was compared with the representation of the same pathway among all the genes on the microarray; −log10 of the Fisher's exact probability is indicated. [score:2]
Figure S10Comparison of transcriptomic changes induced by miR-199a-5p and a siRNA directed against CAV1. [score:2]
Finally, dysregulation of miR-199a-5p was also found in two other mouse mo dels of tissue fibrosis, namely kidney fibrosis and liver fibrosis, suggesting therefore that miR-199a-5p is likely to be a common mediator of fibrosis. [score:2]
In brief, pulmonary fibroblasts were transfected either with pre-miR-199a-5p or negative control as described above. [score:1]
While the highest scoring pathway for miR-199a-5p corresponded to the metabolic pathways “Biosynthesis of Steroids”, we also noticed enrichment for pathways related to “Integrin Signaling” and “Caveolar -mediated Endocytosis Signaling”. [score:1]
We then fused part of the human CAV1 3′UTR to a luciferase reporter using the psiCHEK-2 vector and transfected it into HEK293 cells in the presence of either a pre-miR-199a-5p mimic or a pre-miR-control (Figure 3B). [score:1]
MiR-199a-5p is an effector of TGFβ signaling in lung fibroblasts by regulating CAV1. [score:1]
In the context of tissue fibrosis, both mature forms of miR-199a (i. e., miR-199a-5p and miR-199a-3p) have been associated with the progression of liver fibrosis in both humans and mice [48], [49]. [score:1]
Mutated nucleotides located in the miR-199a-5p -binding site are underlined. [score:1]
MiR-199a is an evolutionary conserved small RNA initially identified in the context of inner ear hair cells development and chondrogenesis [42]– [44] and numerous reports have now shown its implication in various tumor types [45]– [47]. [score:1]
As a control, we also used a CAV1 3′UTR construct mutated on the predicted miR-199a-5p site. [score:1]
These findings strongly suggested that miR-199a-5p may play an important role during the lung fibrosis process. [score:1]
MiR-199a-5p and miR-21 exert indeed similar pro-fibrotic effects on lung fibroblasts. [score:1]
Proposed mo del of miR-199a-5p profibrotic function in lung fibrogenesis. [score:1]
Human pre-miR-199a-5p induced a significant decrease in the normalized luciferase activity relative to control in the presence of the wild type construction only, confirming that it represents a functional site. [score:1]
MiR-199a-5p thus regulates multiple signaling pathways involved in lung fibrogenesis. [score:1]
Cells were serum starved the next day and transfected with pre-miR-199a-5p at 10 nM. [score:1]
As depicted in Figure 11B, in situ hybridization performed 28 days after surgery (i. e. when the fibrosis is established) showed no detectable signal for miR-199a-5p in normal kidney, whereas the hybridization signal was greatly enhanced throughout the injured kidney in area consistent with (myo)fibroblasts. [score:1]
Graphs show the significance of the enrichment, represented as −log [10] (adjusted p-value), according to the fold enrichment using 2 different prediction tools for all known miRNAs: miR-199a-5p and miR-21 are represented as an open diamond and triangle, respectively. [score:1]
As liver fibrosis can regress after cessation of the triggering injury, even at advanced fibrotic stages [63], the decrease of miR-199a-5p observed during resolution of liver fibrosis sets for the first time a specific miRNA as an important player for orchestrating the molecular events occurring during regression of liver fibrosis. [score:1]
In addition, miR-199a-5p was significantly decreased during regression of experimental CCL [4] -induced liver fibrosis (Figure 10D). [score:1]
MiR-199a-5p is dysregulated in mouse mo dels of kidney and liver fibrosis. [score:1]
Normal human pulmonary fibroblasts hFL1 were transfected with pre-miR-Neg, pre-miR-199a-5p or pre-miR-21 (n = 2). [score:1]
Figure S4 Co-transfection of pre-miR-199a-5p or pre-miR-Neg and human CAV1 3′UTR-derived psiCHECK-2 construct in A549 cells show a significant decrease in normalized luciferase activity 48 h post-transfection. [score:1]
Moreover, our data indicated that miR-199a-3p had distinct effects on lung fibroblasts differentiation than miR-199-5p, as assessed by their different impact on αSMA (Figure S14B and data not shown). [score:1]
Functional annotations of the miR-199a-5p experiments highlighted terms such as “Integrin Signaling” and “Caveolar -mediated Endocytosis Signaling”. [score:1]
1003291.g008 Figure 8(A) Heatmap of significant canonical pathways associated with miR-199a-5p, miR-21 and siCAV1 conditions identified through Ingenuity Pathway Analysis. [score:1]
1003291.g002 Figure 2Normal human pulmonary fibroblasts hFL1 were transfected with pre-miR-Neg, pre-miR-199a-5p or pre-miR-21 (n = 2). [score:1]
In particular, our results identified miR-199a-5p as a new determinant of tissue fibrosis. [score:1]
Functional impact of miR-199a-5p on lung fibroblasts. [score:1]
Finally, transfection of pre-miR-199a-5p into MRC-5 and hFL1 lung fibroblasts led to a significant and specific decrease of CAV1 at both mRNA and protein levels while miR-21 had no significant effect (Figure 3C–3E and Figure S5). [score:1]
Our present data establish stromal cells as the primary source of miR-199a-5p in the injured lungs and also suggests that miR-199a-5p is involved in the profibrotic effects mediated by pulmonary fibroblasts. [score:1]
In situ hybridization of miR-199a-5p was performed using double DIG-labeled LNA probes (Exiqon, Woburn, MA). [score:1]
Pre-miR-199a-5p, pre-miR-21 and control miRNA (miR-Neg # 1) were purchased from Ambion. [score:1]
Figure S6 MiR-199a-5p mediates TGFβ dependent differentiation of lung fibroblast into myofibroblasts through CAV1 regulation. [score:1]
Normal human pulmonary fibroblasts hFL1 were transfected with pre-miR-Neg, pre-miR-199a-5p as well as siCAV1 or a control siRNA (n = 2). [score:1]
Lung fibroblasts were transfected with 10 nM of miR-199a-5p mimic or control. [score:1]
HEK293 cells were plated into 96-well and cotransfected using lipofectamin 2000 (Invitrogen) with 0.2 µg of psiCHECK-2 plasmid construct and pre-miR-199a-5p or control miRNA at different concentrations. [score:1]
Pathways that were specific to miR-199a-5p were related to inflammation, such as “IL-1 Signaling”, “Acute Phase Response Signaling” and “P38 MAPK Signaling”, i. e. all typical of fibrotic processes. [score:1]
In this study, we identified a particular miRNA: miR-199a-5p that governs lung fibroblast activation and ultimately lung fibrosis. [score:1]
miR-199a-5p is marked in red. [score:1]
2 logarithm (base 2) of the ratio of miR-199a-5p/miR-Neg (logFC). [score:1]
A combination of in silico and experimental data, described in [25], [26], [40], identified the transcripts affected by miR-199a-5p in lung fibroblasts. [score:1]
Data were treated according to Gene Set Enrichment Analysis (GSEA) software, with transcripts being ordered according to miR-199a-5p versus miR-Neg conditions scaled by their standard deviation. [score:1]
The linear fold ratio for CAV1 between IPF and control was 0.54 (FDR<0.05) and the linear fold ratio for miR-199a-5p for the same subjects was 1.35 (p<0.05). [score:1]
Lung fibroblasts were transfected by miR-199a-5p mimic, si-CAV1 or negative controls. [score:1]
The mechanisms involved in the TGFβ -dependent modulation of miR-21 and miR-199a-5p are also of particular interest. [score:1]
Among several miRNAs candidates with such a profile, miR-199a-5p displayed the highest statistical score (Figure 1B). [score:1]
MiR-199a-5p is commonly dysregulated in three experimental mouse mo dels of liver, lung, and kidney fibrosis. [score:1]
Alignment of miR-199a-5p with human CAV1 3′UTR sequence revealed one potential conserved seed site (Figure 3A). [score:1]
In situ hybridization In situ hybridization of miR-199a-5p was performed using double DIG-labeled LNA probes (Exiqon, Woburn, MA). [score:1]
Transfection of miR-199a-5p precursors resulted in a significant induction of migration (Figure 7A and 7B) and invasion (Figure 7C). [score:1]
Figure S15 Transfection of human lung fibroblasts with pre-miR-199a-5p or pre-miR-21 increases cell motility. [score:1]
miR-199a-5p is an effector of the TGFβ pathway. [score:1]
Figure S16 Effect of TGFβ on mature and pri-miRNAs forms of miR-199a in human lung fibroblasts. [score:1]
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[+] score: 414
Other miRNAs from this paper: mmu-mir-199a-1
Ad-anti-miR199a-5p transcribes reverse miR199a-5p sequences from the vector to silence the expression of miR199a-5p, Ad-anti-miR NC expresses Ad-anti-miR199a-5p’s control scrambled short hairpin RNA, Ad-miR199a-5p overexpresses miR199a-5p, Ad-miR NC expresses Ad-miR199a-5p’s control scrambled short hairpin RNA, Ad-ATG14 overexpresses ATG14, and Ad-NC is Ad-ATG14’s control and expresses green fluorescent protein. [score:13]
miRNAs have been much appreciated as major regulators of gene expression with crucial functions in numerous diseases, including neurological disorders, heart diseases, vascular diseases, viral infections and cancers 4, 5. Recent studies have identified several miRNAs that are associated with the core components of metabolic diseases [6], such as insulin resistance and lipid metabolism 7– 9. Among these miRNAs, miR199a-5p, which can be produced from two genes, miR199a-1 and miR199a-2, is an interesting miRNA that has been shown to contribute to lung fibrotic processes, embryonic stem cell-derived hepatic cell repopulation and liver cancer glucose metabolism 10– 13. [score:12]
Among these nine hits, Cav1 and Atg14 were significantly upregulated in mice not expressing miR199a-5p (Fig.   1a), whereas they were downregulated in mice overexpressing miR199a-5p (Fig.   1b). [score:11]
Furthermore, we uncovered a novel mechanism whereby dysregulated miR199a-5p suppresses the expression of ATG14 and inhibits autophagy, which in turn leads to the downregulation of hepatic insulin sensitivity and eventually causes insulin resistance (Fig.   7h). [score:11]
To fully uncover the molecular mechanism by which miR199a-5p regulates insulin sensitivity, we profiled the expression levels of potential target genes of miR199a-5p, which had been reported as target genes of miR199a-5p or as being associated with glucose or lipid metabolism 9, 22, 32– 35, and identified ATG14 as a target of miR199a-5p. [score:10]
In the current study, we also found that hepatic CAV1 was upregulated or downregulated in miR199a-5p knocked-down or overexpressing mice, respectively. [score:10]
Fig. 6 a HepG2 cells and primary hepatocytes were infected with adenovirus -expressing miR199a-5p (Ad-anti-miR199a-5p) or the negative control (Ad-anti-miR NC) for 48 h. b HepG2 cells and primary hepatocytes were infected with adenovirus -expressing miR199a-5p (Ad-miR199a-5p) or the negative control (Ad-miR NC) for 48 h. c Primary hepatocytes were infected with adenovirus -expressing ATG14 (Ad-ATG14) or the negative control (Ad-NC) for 48 h. d Primary hepatocytes were infected with adenoviruses expressing miR199a-5p (Ad-miR199a-5p) and ATG14 (Ad-ATG14) or the negative control (Ad-NC) for 48 h, and then treated with 5 μM 3-MA for 2 h. All cases were followed with or without 100 nmol/L insulin (Ins) stimulation for 20 min. [score:9]
Data shown are representative of three independent experiments a HepG2 cells and primary hepatocytes were infected with adenovirus -expressing miR199a-5p (Ad-anti-miR199a-5p) or the negative control (Ad-anti-miR NC) for 48 h. b HepG2 cells and primary hepatocytes were infected with adenovirus -expressing miR199a-5p (Ad-miR199a-5p) or the negative control (Ad-miR NC) for 48 h. c Primary hepatocytes were infected with adenovirus -expressing ATG14 (Ad-ATG14) or the negative control (Ad-NC) for 48 h. d Primary hepatocytes were infected with adenoviruses expressing miR199a-5p (Ad-miR199a-5p) and ATG14 (Ad-ATG14) or the negative control (Ad-NC) for 48 h, and then treated with 5 μM 3-MA for 2 h. All cases were followed with or without 100 nmol/L insulin (Ins) stimulation for 20 min. [score:9]
Zhang B Upregulated microRNA-199a-5p inhibits nuclear receptor corepressor 1 translation in mice with nonalcoholic steatohepatitisMol. [score:8]
More importantly, we demonstrated that in type 2 diabetes patients and animal mo dels the hepatic miR199a-5p level is upregulated, whereas the ATG14 level and autophagy levels are suppressed, which implies the importance of the miR199a-5p/ATG14 axis in translational diabetes research. [score:8]
b The expression profiles of potential targets of miR199a-5p in male C57BL/6J WT mice overexpressing miR199a-5p. [score:7]
We propose that miR199a-5p inhibits ATG14 -mediated autophagy, suppresses hepatic insulin sensitivity and eventually leads to insulin resistance, which is a key diabetogenic factor To study the effect of miR199a-5p on insulin sensitivit y, we first injected Ad-anti-miR199a-5p adenoviruses or control scrambled adenoviruses (Ad-anti-miR NC) into male HFD-fed C57BL/6J WT mice through the tail vein and then checked the expression of miR199a-5p. [score:7]
Our study also demonstrated the significance of the miR199a-5p/ATG14 axis in translational diabetes research, which may serve as a potential therapeutic target for hepatic insulin sensitivity and other associated metabolic diseases. [score:7]
Fig. 5 a- c HepG2 cells and primary hepatocytes that originated from male HFD-fed C57BL/6J WT mice were infected with adenovirus-inhibiting miR199a-5p (Ad-anti-miR199a-5p) or the negative control (Ad-anti-miR NC) for 48 h. d-f HepG2 cells and primary hepatocytes that originated from male C57BL/6J WT mice were infected with adenovirus -expressing miR199a-5p (Ad-miR199a-5p) or the negative control (Ad-miR NC) for 48 h. The expression level of miR199a-5p and the protein levels of ATG14, BECLIN1 and LC3 in HepG2 cells and primary hepatocytes are shown in a, b and d, e, respectively. [score:7]
a- c HepG2 cells and primary hepatocytes that originated from male HFD-fed C57BL/6J WT mice were infected with adenovirus-inhibiting miR199a-5p (Ad-anti-miR199a-5p) or the negative control (Ad-anti-miR NC) for 48 h. d-f HepG2 cells and primary hepatocytes that originated from male C57BL/6J WT mice were infected with adenovirus -expressing miR199a-5p (Ad-miR199a-5p) or the negative control (Ad-miR NC) for 48 h. The expression level of miR199a-5p and the protein levels of ATG14, BECLIN1 and LC3 in HepG2 cells and primary hepatocytes are shown in a, b and d, e, respectively. [score:7]
The two-tailed Student's t-test was applied to determine the difference between the psiCHECK2 group and the WT 3ʹ-UTR group (** P < 0.01) or the psiCHECK2 group and the Mut 3ʹ-UTR group (NS, P > 0.05) a The expression profiles of potential targets of miR199a-5p in male HFD-fed C57BL/6J WT mice expressing miR199a-5p. [score:7]
Fig. 1 a The expression profiles of potential targets of miR199a-5p in male HFD-fed C57BL/6J WT mice expressing miR199a-5p. [score:7]
Lino Cardenas CL miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta -induced lung fibroblast activation by targeting caveolin-1PLoS Genet. [score:6]
To validate the involvement of these genes, their mRNA expression profiles were examined in mice with knocked-down or overexpressed miR199a-5p. [score:6]
We also detected upregulated miR199a-5p (Fig.   7e) and suppressed Atg14 mRNA levels (Fig.   7f) in these patients, which was consistent with our hypothesis. [score:6]
miR199a-1 and miR199a-2 are precursors for miR199a-5p, and evolutionary studies have found that the predominant expression patterns of miR199a-1 and miR199a-2 in tissues surrounding developing craniofacial skeletal elements are consistent between humans and mice, indicating a conserved role of miR199a-1 and miR199a-2 in vertebrate skeletogenesis [38] and implying the potential translational application of miR199a-5p from mice to humans in the future. [score:5]
We propose that miR199a-5p inhibits ATG14 -mediated autophagy, suppresses hepatic insulin sensitivity and eventually leads to insulin resistance, which is a key diabetogenic factor a- d The levels of fasting glucose, HbA1c, fasting insulin and HOMA-IR in diabetes patients. [score:5]
To further identify the downstream effector of miR199a-5p, we first predicted potential miR199a-5p mRNA targets by TargetScan, an online bioinformatics tool, and then filtered by conserved binding sites on 8-mer, 7-mer-m8 or 7-mer-A1. [score:5]
The two-tailed Student's t-test was applied to determine the difference between the Ad-anti-miR199a-5p group or the Ad-miR199a-5p group and the corresponding control group (** P < 0.01) a ATG14, BECLIN1 and LC3 expression levels in male HFD-fed C57BL/6J WT mice expressing miR199a-5p. [score:5]
We observed that the luciferase activity was significantly suppressed in the group co -transfected with WT Atg14 and miR199a-5p, but not the group co -transfected with the mutated Atg14 3ʹ-UTR reporter and Ad-miR199a-5p (Fig.   1d), suggesting that Atg14 is a target of miR199a-5p. [score:5]
Fig. 3. a ATG14, BECLIN1 and LC3 expression levels in male HFD-fed C57BL/6J WT mice expressing miR199a-5p. [score:5]
miR199a-5p inhibits insulin sensitivity by suppressing ATG14 in vitro. [score:5]
Furthermore, electron microscopic examination of liver samples demonstrated significant increases or decreases in autophagosome/autolysosome formation in mice with inhibited or overexpressed miR199a-5p (Figs.   3c-f), indicating a link between miR199a-5p and autophagy. [score:5]
As shown in Supplementary Fig.   1A-B, miR199a-5p had no effect on gluconeogenesis in the liver, the expression of G6Pase or the expression of PEPCK, implying that the effect of miR199a-5p mainly occurred through improved insulin sensitivity rather than gluconeogenesis. [score:5]
To gain more insights into the importance of ATG14 in the regulation of insulin sensitivity, we infected primary hepatocytes with Ad-ATG14 viruses to cause the overexpression of ATG14 and found that insulin -induced phosphorylation of IRβ, AKT and GSK3β was significantly decreased in those cells (Fig.   6c), supporting the idea that miR199a-5p regulates insulin sensitivity through ATG14 in vitro. [score:5]
We first examined the effects of Ad-miR199a-5p on ATG14 protein levels in mice, and found that the ATG14 expression levels were decreased by Ad-miR199a-5p (Fig.   4a) but could be partially rescued by the overexpression of Ad-ATG14 (Fig.   4a). [score:5]
We propose that miR199a-5p inhibits ATG14 -mediated autophagy, suppresses hepatic insulin sensitivity and eventually leads to insulin resistance, which is a key diabetogenic factor It has been reported that miR199a-5p contributes to lung fibrotic processes, embryonic stem cell-derived hepatic cell repopulation, liver cancer glucose metabolism 10– 13 and hepatic steatosis [14]. [score:5]
The two-tailed Student's t-test was applied to determine the difference between the diabetes group and the control group, * P < 0.05, ** P < 0.01, t-test, n = 8. h Schematic mo del in which miR199a-5p inhibits hepatic insulin sensitivity via suppressing ATG14 -mediated autophagy. [score:5]
Overexpressed miR199a-5p leads to insulin sensitivity inhibition in vivo. [score:5]
b ATG14, BECLIN1 and LC3 expression levels in male C57BL/6J WT mice overexpressing miR199a-5p. [score:5]
The two-tailed Student's t-test was applied to determine the difference between the Ad-anti-miR199a-5p group or the Ad-miR199a-5p group and the corresponding control group (** P < 0.01, *** P < 0.001) On the other hand, after overexpressing miR199a-5p in HepG2 cells or primary hepatocytes by Ad-miR199a-5p viruses (Figs.   5d, e), we detected lower protein levels of ATG14, BECLIN1 and LC3 (Figs.   5d, e) and decreased autophagic flux in primary hepatocytes originating from male C57BL/6J WT mice (Fig.   5f), suggesting that miR199a-5p regulates autophagy and autophagic flux via ATG14. [score:4]
Our data supported that ATG14 is a direct target of miR199a-5p and that its role in the miR199a-5p cascade caused insulin resistance. [score:4]
Second, knocking down miR199a-5p led to decreased insulin -induced phosphorylation of IRβ, AKT and GSK3β in vitro, and overexpression of miR199a-5p potentiated their phosphorylation. [score:4]
miR199a-5p regulates insulin sensitivity by targeting ATG14. [score:4]
We first showed that knocking down miR199a-5p resulted in decreased glucose tolerance and clearance in vivo and that overexpression of miR199a-5p had the opposite effect. [score:4]
Among the known autophagy-related proteins, autophagy-related protein 14 (ATG14) is the most important regulator involved in autophagic initiation [22], and we found that ATG14 is an interesting target of miR199a-5p. [score:4]
Because gluconeogenesis is another critical step in regulating hepatic glucose production [28], we assessed by PTT assay whether miR199a-5p affected gluconeogenesis and checked the expression levels of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) [29], two key enzymes involved in regulating gluconeogenesis. [score:4]
In HepG2 cells and primary hepatocytes with knocked-down or overexpressed miR199a-5p, the phosphorylation of the three key components of insulin signaling was significantly decreased or increased, respectively (Figs.   6a, b), indicating improved and diminished insulin sensitivity, respectively. [score:4]
The two-tailed Student's t-test was applied to determine the difference between the Ad-anti-miR199a-5p group or the Ad-miR199a-5p group and the corresponding control group (** P < 0.01, *** P < 0.001)On the other hand, after overexpressing miR199a-5p in HepG2 cells or primary hepatocytes by Ad-miR199a-5p viruses (Figs.   5d, e), we detected lower protein levels of ATG14, BECLIN1 and LC3 (Figs.   5d, e) and decreased autophagic flux in primary hepatocytes originating from male C57BL/6J WT mice (Fig.   5f), suggesting that miR199a-5p regulates autophagy and autophagic flux via ATG14. [score:4]
We further filtered these hits based on the following criteria: genes that were reported as target genes of miR199a-5p or genes that were reported as being associated with glucose or lipid metabolism. [score:3]
Data shown are representative of three independent experiments Finally, we explored the clinical relevance of miR199a-5p by examining miR199a-5p expression levels in liver samples derived from diabetes patients. [score:3]
ATG14 is miR199a-5p’s target. [score:3]
Mature miR199a-5p is a molecule 21 nucleotides in length with several proteins as downstream targets 14, 30, 31. [score:3]
e, f The expression levels of miR199a-5p and Atg14. [score:3]
The decreased fasting blood glucose and insulin levels in the Ad-anti-miR199a-5p group resulted in a lower HOMA-IR index (Fig.   2d), suggesting that knocking down miR199a-5p improved insulin sensitivity, a key factor in regulating glucose homeostasis. [score:3]
d Representative electron microscopic images in male C57BL/6J WT mice overexpressing miR199a-5p. [score:3]
c Representative electron microscopic images in male HFD-fed C57BL/6J WT mice expressing miR199a-5p. [score:3]
Similarly, the PTT data and the G6Pase and PEPCK gene expression data indicated that miR199a-5p did not affect gluconeogenesis in the liver (Supplementary Fig.   1C-D). [score:3]
The two-tailed Student's t-test was applied to determine the difference between the Ad-miR199a-5p group and the control group (* P < 0.05, ** P < 0.01, *** P < 0.001), n = 6 mice per group a- e Male C57BL/6J WT mice were fed a high-fat diet for 3 months and then infected with adenovirus-inhibiting miR199a-5p (Ad-anti-miR199a-5p) or the negative control (Ad-anti-miR NC) via tail vein injection. [score:3]
f– j Male C57BL/6J WT mice fed a chow diet were infected with adenovirus -expressing miR199a-5p (Ad-miR199a-5p) or the negative control (Ad-miR NC) via tail vein injection. [score:3]
The expression profile of miR199a-5p in the livers of diabetes patients. [score:3]
Using this approach, nine genes were included in our list: Cav1, Hif1α and Ncor1 were reported as target genes of miR199a-5p; 14, 30, 31 Tab3, Atg14, Sirt1, Grb10, Map3k5 and Ucp3 were reported as being associated with glucose or lipid metabolism 9, 22, 32– 35. [score:3]
Although the overexpression of Ad-ATG14 had no effects on the fed blood glucose or serum insulin levels (Figs.   4b, c), it increased the depressed levels of fasting blood glucose, fasting serum insulin and the HOMA-IR index caused by Ad-miR199a-5p back to normal levels (Figs.   4b–d). [score:3]
We then focused on Atg14 because a more robust change in the expression level was detected for Atg14 than for Cav1 when the miR199a-5p level was manipulated. [score:3]
In addition, Ad-ATG14 markedly inhibited miR199a-5p-potentiated glucose and insulin tolerance (Fig.   4e). [score:3]
To further assess the effect of miR199a-5p on the dynamics of autophagy, we co-infected primary hepatocytes originating from male HFD-fed C57BL/6J WT mice with adenoviruses expressing mCherry and GFP double-tagged LC3 and Ad-anti-miR199a-5p. [score:3]
Fig. 2 a- e Male C57BL/6J WT mice were fed a high-fat diet for 3 months and then infected with adenovirus-inhibiting miR199a-5p (Ad-anti-miR199a-5p) or the negative control (Ad-anti-miR NC) via tail vein injection. [score:3]
To further validate Atg14 as a target of miR199a-5p, we co -transfected the psiCHECK2-promoter -based Atg14 3ʹ-UTR reporter with Ad-miR199a-5p or the negative control into HEK293T cells. [score:3]
Consistently, all three proteins’ levels were decreased in miR199a-5p -overexpressing mice (Fig.   3b). [score:3]
Inhibition of miR199a-5p by Ad-anti-miR199a-5p increases insulin sensitivity in vivo. [score:3]
To study the effect of miR199a-5p on insulin sensitivit y, we first injected Ad-anti-miR199a-5p adenoviruses or control scrambled adenoviruses (Ad-anti-miR NC) into male HFD-fed C57BL/6J WT mice through the tail vein and then checked the expression of miR199a-5p. [score:3]
To analyze the expression of miR199a-5p, we used gene-specific TaqMan miRNA assay probes (Applied Biosystems, Foster City, CA). [score:2]
Mobus S MicroRNA-199a-5p inhibition enhances the liver repopulation ability of human embryonic stem cell-derived hepatic cellsJ. [score:2]
Because of liver’s essential role in regulating lipid and glucose metabolism and a potential link between hepatic insulin resistance and hepatic steatosis 15, 16, we hypothesized that miR199a-5p plays a key role in hepatic insulin sensitivity. [score:2]
Our previous work found that miR199a-5p/hepatic steatosis could be regulated by free fatty acids (FFAs) [14], and other reports have also suggested a potential link between hepatic insulin resistance and hepatic steatosis 15, 16. [score:2]
The two-tailed Student's t-test was applied to determine the difference between the Ad-anti-miR199a-5p group or the Ad-miR199a-5p group and the corresponding control group (** P < 0.01, *** P < 0.001) To explore the role of miR199a-5p in the regulation of insulin sensitivity in vitro, we assessed insulin signaling by examining insulin-stimulated phosphorylation of IRβ (Tyr1162/1163), AKT (Ser473) and GSK3β (Ser9) [36]. [score:2]
To our knowledge, this study is the first report to illustrate a novel biologic function of miR199a-5p in the regulation of hepatic insulin sensitivity. [score:2]
The two-tailed Student's t-test was applied to determine the difference between the Ad-anti-miR199a-5p group or the Ad-miR199a-5p group and the corresponding control group (** P < 0.01) To gain more insights into the connection between ATG14 and miR199a-5p in the regulation of insulin sensitivity, we injected mice with Ad-miR199a-5p with or without Ad-ATG14. [score:2]
miR199a-5p regulates insulin sensitivity in vivo. [score:2]
Because hepatic insulin resistance is closely related to fatty liver, we tested the hypothesis that miR199a-5p regulates hepatic insulin sensitivity 15, 16. [score:2]
To explore the role of miR199a-5p in the regulation of insulin sensitivity in vitro, we assessed insulin signaling by examining insulin-stimulated phosphorylation of IRβ (Tyr1162/1163), AKT (Ser473) and GSK3β (Ser9) [36]. [score:2]
Compared with the level in mice that were infected with the control Ad-anti-miR NC viruses, the hepatic miR199a-5p level was significantly inhibited in mice infected with Ad-anti-miR199a-5p viruses, which reduced the miR199a-5p level (Fig.   2a). [score:2]
miR199a-5p regulates autophagy in vitro. [score:2]
The two-tailed Student's t-test was applied to determine the difference between the Ad-ATG14 group and the corresponding control group (* P < 0.05, ** P < 0.01), n = 8 mice per group To explore the regulation of autophagy by miR199a-5p in vitro, we infected HepG2 cells or primary hepatocytes with Ad-anti-miR199a-5p viruses and found that the miR199a-5p level was decreased in these cells. [score:2]
We found that the protein levels of ATG14, BECLIN1 and LC3 were increased in HFD mice with knocked-down miR199a-5p (Fig.   3a). [score:2]
miR199a-5p regulates autophagy and autophagic flux in vitro. [score:2]
miR199a-5p regulates autophagy in vivo. [score:2]
In conclusion, our data provide strong evidence to support the role of miR199a-5p in the regulation of hepatic insulin sensitivity. [score:2]
However, we cannot rule out the possibility that the effect of miR199a-5p on the regulation of hepatic insulin sensitivity may be partially mediated by CAV1 in mice. [score:2]
d HEK cells were co -transfected with the psiCHECK2-promoter vector and the WT 3ʹ-UTR report plasmid of ATG14 or the mutated 3ʹ-UTR report plasmids of ATG14 (Mut ATG14) with miR199a-5p (Ad-miR199a-5p) or the negative control (Ad-miR NC), and the luciferase signal was determined. [score:1]
The two-tailed Student's t-test was applied to determine the difference between the group receiving Ad-anti-miR199a-5p and the control group (* P < 0.05, ** P < 0.01, *** P < 0.001), n = 8 mice per group. [score:1]
The two-tailed Student's t-test was applied to determine the difference between the Ad-miR199a-5p group and the corresponding control or the Ad-anti-miR199a-5p group and the corresponding control (* P < 0.05, *** P < 0.001), n = 6-8. c Sequence alignment of miR199a-5p within the 3ʹ-UTRs of human and mouse ATG14. [score:1]
Interestingly, ATG14, BECLIN1 and LC3 protein levels were increased (Figs.   5a, b), highlighting the association between miR199a-5p and the autophagy pathway. [score:1]
After being seeded into 24-well plates, HEK293T cells were co -transfected with psiCHECK2 plasmids and Ad-miR199a-5p or the control plasmid using lipofectamine 2000 (Invitrogen). [score:1]
We then found that the potential miR199a-5p binding site within Atg14 is highly conserved between humans, mice (Fig.   1c) and other eutherians. [score:1]
The two-tailed Student's t-test was applied to determine the difference between the Ad-ATG14 group and the corresponding control group (* P < 0.05, ** P < 0.01), n = 8 mice per group a– e Male C57BL/6J WT mice were infected with Ad-miR199a-5p (Ad-miR199a-5p) or the negative control (Ad-miR NC) and with Ad-ATG14 (Ad-ATG14) or the negative control (Ad-NC) via tail vein injection. [score:1]
The seed region of miR199a-5p is GUGACC. [score:1]
For mouse miR199a-2, the miRBase accession number is MI0000713, and the MGI ID is 3618742. [score:1]
Fig. 4 a– e Male C57BL/6J WT mice were infected with Ad-miR199a-5p (Ad-miR199a-5p) or the negative control (Ad-miR NC) and with Ad-ATG14 (Ad-ATG14) or the negative control (Ad-NC) via tail vein injection. [score:1]
The miR199a-5p sequence is highly conserved in humans and mice. [score:1]
miR199a-5p induces insulin resistance by decreasing ATG14 in vivo. [score:1]
Moreover, our previous study demonstrated that miR199a-5p is involved in hepatic steatosis [14]. [score:1]
miR199a-5p leads to hepatic insulin resistance via ATG14 in vivo. [score:1]
Furthermore, TEM data demonstrated a significant reduction in autophagosome/autolysosome formation in diabetes patients (Fig.   7g), strongly supporting a correlation between miR199a-5p/ATG14 -mediated autophagy and hepatic insulin sensitivity in diabetes patients. [score:1]
As shown in Fig.   5c, Ad-anti-miR199a-5p promoted a marked increase in autolysosomes, which was consistent with western blot data (Figs.   5a, b). [score:1]
The annealed oligonucleotides with the WT ATG14 3ʹ-UTR binding site or the oligonucleotides with mutated miR199a-5p -binding sites (Fig.   1c) were cloned into psiCHECK2 reporter vectors (Promega) between the XhoI and NotI sites. [score:1]
f Day 9: measurement of hepatic miR199a-5p expression. [score:1]
The two-tailed Student's t-test was applied to determine the difference between the Ad-miR199a-5p group and the control group (* P < 0.05, ** P < 0.01, *** P < 0.001), n = 6 mice per group To further explore the role of miR199a-5p in insulin sensitivity, we tried another approach by injecting Ad-miR199a-5p adenoviruses or its control scrambled adenoviruses (Ad-scrambled) into male C57BL/6J WT mice fed normal chow, and detected a significantly higher miR199a-5p level in the mice infected with Ad-miR199a-5p viruses than in mice infected with control viruses (Fig.   2f). [score:1]
Li B Aberrant miR199a-5p/caveolin1/PPARalpha axis in hepatic steatosisJ. [score:1]
Importantly, we provided evidence of increased hepatic miR199a-5p levels in diabetes patients, which correlated with decreased ATG14 levels and autophagy. [score:1]
Aberrant miR199a-5p/ATG14/hepatic insulin sensitivity axis in diabetes patients. [score:1]
Zhang PX Pharmacological modulation of the AKT/microRNA-199a-5p/CAV1 pathway ameliorates cystic fibrosis lung hyper-inflammationNat. [score:1]
Although the serum insulin level under the fed condition was not affected by Ad-anti-miR199a-5p, a decrease in serum insulin level was observed under the fasting condition in the Ad-anti-miR199a-5p group (Fig.   2c). [score:1]
In comparison with the levels in the control group, the fasting and fed blood glucose levels were significantly lower in Ad-anti-miR199a-5p mice (Fig.   2b). [score:1]
miR199a-5p leads to insulin resistance via ATG14 in vitro. [score:1]
Therefore, we further proposed a miR199a-5p/ATG14/autophagy/insulin resistance axis in diabetes pathogenesis in our study. [score:1]
The miRBase accession number for mouse miR199a-5p is MIMAT0000229, and the miRBase accession number of human miR199a-5p is MIMAT0000231. [score:1]
A higher HOMA-IR index in Ad-miR199a-5p mice was also observed due to the elevated fasting blood glucose and insulin levels (Fig.   2i). [score:1]
We further examined GTTs and ITTs and found that Ad-anti-miR199a-5p mice exhibited reduced glucose tolerance and clearance (Fig.   2e). [score:1]
The two-tailed Student's t-test was applied to determine the difference between the Ad-miR199a-5p group and the corresponding control group (* P < 0.05, ** P < 0.01, *** P < 0.001) or between the Ad-miR199a-5p group with Ad-ATG14 and the Ad-miR199a-5p group without Ad-ATG14. [score:1]
An increase in the serum insulin level was observed under the fasting condition with miR199a-5p, but not under the fed condition (Fig.   2h). [score:1]
a Day 9: measurement of hepatic miR199a-5p expression. [score:1]
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[+] score: 383
Consistent with c-MYC representing a functional downstream target of miR-199a-3p, knocking down endogenous c-MYC using 3 individual c-MYC -targeting siRNAs (Supplementary Figure 3B) or inhibiting c-MYC expression using JQ1 [36] (Supplementary Figure 3C) both inhibited PC3 cell viability (Figure 7C-7D). [score:12]
Interestingly, miR-199a-3p overexpression did not reduce CD44 mRNA levels in PCa cells (Figure 6D), suggesting that miR-199a-3p likely targets CD44 in PCa cells by causing translational inhibition. [score:9]
Of note, either overexpression (data not sown) or knockdown of c-MYC (Supplementary Figure 3E–3F) had no effect on miR-199a-3p expression, although c-MYC was reported to modulate the expression of a number of miRNAs involved in the cell cycle and apoptosis [11, 37]. [score:8]
To test this possibility, we employed 8 different target-prediction programs, 3 of which (i. e., RNA22, TargetMiner, and TargetScan) simultaneously identified a putative binding site of miR-199a-3p at the CD44 3′-UTR (Figure 6A-6B). [score:7]
These discussions, together with the fact that miR-199a-3p was significantly underexpressed in CD44 [+] PCa cells [15] (Figure 1A), raise the possibility that miR-199a-3p exerts its PCa -inhibitory effects via targeting, at least partly, CD44. [score:7]
Regardless, by simultaneously targeting a cohort of pro-oncogenic molecules, miR-199a-3p manifests powerful PCa-suppressing effects, mainly through inhibiting cell proliferation (Figure 7I). [score:7]
Since our preceding experiments have shown that miR-199a-3p suppressed PCa primarily by inhibiting cell-cycle progression and cell proliferation, we subsequently focused our efforts on 3 mitogenic molecules important for regulating PCa cell proliferation, i. e., c-MYC, cyclin D1, and EGFR. [score:6]
The fact that 3 tumor-suppressive miRNAs, i. e., miR-34a [9], miR-708 [28], and miR-199a-3p (this study), simultaneously target 5 different sites at the CD44 3′-UTR (Figure 6A), highlights the critical importance of CD44 in regulating CSC properties [6– 10]. [score:6]
Mechanistically, we find that like miR-34a, which is also under-expressed in CD44 [+] PCSCs [9], miR-199a-3p directly targets CD44 in several PCa cell types. [score:6]
We have shown that overexpression of miR-199a-3p greatly inhibits proliferation and clonal and sphere-forming capacities of CD44 [+] as well as bulk PCa cells. [score:5]
Expression of miR-199a-3p inhibits cell proliferation. [score:5]
Notably, our present study has provided evidence that miR-199a-3p may also exert tumor-suppressive functions via modulating several novel targets, i. e., c-MYC, cyclin D1, and EGFR. [score:5]
Notably, miR-199a-3p inhibited secondary sphere formation in PC3 cells (Figure 2F), suggesting that miR-199a-3p may inhibit PCSC self-renewal in vitro. [score:5]
Taken together, the above experiments indicate that miR-199a-3p inhibits prostate tumor regeneration and growth by inhibiting cell proliferation without causing cell death. [score:5]
These results indicate that reduced expression of c-MYC facilitates the inhibitory effect of miR-199a-3p in PCa cells such as PC3. [score:5]
Taken together, these observations indicate that enforced expression of miR-199a-3p inhibits PCa cell cell-cycle progression and proliferation without affecting cell death or senescence. [score:5]
Impressively, miR-199a-3p expression inhibits both tumor initiation and tumor growth in several PCa xenograft mo dels. [score:5]
miR-199a-3p suppresses prostate tumor regeneration in vivomiR-199a-3p has been shown to inhibit peritoneal dissemination of ovarian carcinoma cells in a xenograft mo del [23]. [score:5]
Strikingly, miR-199a-3p overexpression completely inhibited tumor regeneration from bulk DU145 cell (Figure 4D, b). [score:5]
miR-199a-3p inhibits PCa cell proliferation in vitromiR-199a-3p has been reported as a tumor suppressive miRNA in several tumor types. [score:5]
miR-199a-3p overexpression by oligo transfection also inhibited tumor regeneration in PPC-1 and PC3 cells (data not shown). [score:5]
At 10,000 injections, miR-199a-3p inhibited both tumor incidence and tumor growth (Figure 4A; note that miR-199a-3p overexpressing CD44 [+]DU145 cells regenerated tumors that were only 1/10 of the tumors derived from NC -transfected CD44 [+]DU145 cells). [score:5]
We also show that miR-199a-3p exerts its PCa suppressive functions via targeting CD44 and several mitogenic molecules including c-MYC, cyclin D1 and EGFR. [score:5]
We wondered what other molecules miR-199a-3p might also target in PCa cells, either directly or indirectly. [score:5]
In contrast, mutations in the miR-199a-3p binding site at CD44 3′-UTR abolished the luciferase -inhibitory effects of miR-199a-3p in both cell types (Figure 6C). [score:4]
These results indicate that miR-199a-3p regulates cyclin D1 and EGFR expression in PC3 cells. [score:4]
Of note, miR-199a-3p downregulated exogenous c-MYC protein derived from a c-MYC-encoding cDNA construct in both PC3 and DU145 cells (Figure 7A; lanes 6 and 10 vs. [score:4]
CD44 is a direct target of miR-199a-3p. [score:4]
Kinose et al reported that miR-199a-3p was downregulated under hypoxia and decreased the clonal capacity in ovarian cancer cells [23]. [score:4]
The results revealed significant under -expression of miR-199a-3p in all three CD44 [+] PCa cell populations (Figure 1A). [score:3]
To explore the therapeutic potential of miR-199a-3p in PCa, we set out to test its tumor -inhibitory effects in a pre-established PCa xenograft mo del. [score:3]
Finally, miR-199a-3p was reported to target CD44 in HCC cells [19]. [score:3]
miR-199a-3p suppresses prostate tumor regeneration in vivo. [score:3]
Finally, consistent with previous reports that miR-199a-3p also targets other oncogenic molecules such as mTOR [18, 20], we observed reduced mTOR protein levels in DU145 cells transfected with miR-199a-3p oligos (Figure 7H). [score:3]
C. Schematic showing miR-199a-3p expressing vector pGIPZ-199A based on GIPZ lentiviral shRNA backbone (pGIPZ-Ctrl). [score:3]
miR-199a-3p inhibits clonal and clonogenic properties of HPCa cells. [score:3]
To uncover the potential mechanisms underlying the PCa cell “growth -inhibitory” effects of miR-199a-3p, we assessed cell proliferation by BrdU incorporation and cell-cycle (i. e., DNA content) analysis, cell death by Annexin V and PI staining, and cell senescence by senescence -associated β-galactosidase staining. [score:3]
miR-199a-3p inhibits PCSC properties. [score:3]
miR-199a-3p also targets c-MYC and several other mitogenic signaling molecules. [score:3]
Figure 5 A. Schematic of inducible miR-199a-3p expressing lentiviral vector. [score:3]
Most miR-199a-3p related studies are in hepatocellular carcinoma (HCC), in which it is reported to induce apoptosis or to suppress cell proliferation by delaying G1/S transition [18– 20]. [score:3]
lanes 5 and 9, respectively), suggesting that miR-199a-3p might target c-MYC coding sequence. [score:3]
We first processed the raw data using the ΔCt method, by which the expression level of miR-199a-3p in each sample was normalized to that of RNU48. [score:3]
I. A schematic summarizing downstream targets of miR-199a-3p in PCa cells. [score:3]
miR-199a-3p has been shown to inhibit peritoneal dissemination of ovarian carcinoma cells in a xenograft mo del [23]. [score:3]
miR-199a-3p has been reported as a tumor suppressive miRNA in several tumor types. [score:3]
In fact, miR-199a-3p has been shown to suppress, in addition to CD44 [19], several other molecules including MET, mTOR, and PAK4 [18, 20]. [score:3]
To that end, we first constructed a doxycycline (Dox) inducible lentiviral system to overexpress miR-199a-3p (lenti-199a), in which primary miR-199A1 sequence was cloned downstream from the RFP reporter (Figure 5A). [score:3]
Bulk HPCa cells with miR-199a-3p overexpression demonstrated much lower sphere-forming (Figure 3A-3B) and clonal (Figure 3C-3D) capacities than the corresponding HPCa cells transfected with NC oligos. [score:3]
Note that the miR-199A1 lentivector did encode miR-199a-5p; however, the miR-199a-5p levels in both DU145 and LAPC9 cells were much lower than miR-199a-3p levels (Figure 4D-4E), suggesting that the PCa-suppressive effects we observed were largely ascribed to miR-199a-3p. [score:3]
It seems that miR-199a-3p may target a different cohort of molecules in different PCa cell types. [score:3]
A. Schematic of inducible miR-199a-3p expressing lentiviral vector. [score:3]
Consistent with the cell-cycle analysis, miR-199a-3p inhibited BrdU incorporation in PC3 (Figure 1I) and DU145 (Figure 1J) cells. [score:3]
In some experiments, bulk or purified CD44 [+] cells were infected with empty (pGIPZ-Ctrl) or miR-199a-3p expressing lentivirus (pGIPZ-199A) at MOI (multiplicity of infection) of 10-20 for 72 h. pGIPZ-199A vector was established from the backbone of GIPZ lentiviral shRNA (GIPZ-Ctrl) (GE Dharmacon), in which pre-miR-199A1 and its frank sequences were cloned into XhoI and MluI sites (Supplementary Figure 1A). [score:3]
miR-199a-3p inhibits PCa cell proliferation in vitro. [score:3]
So how did miR-199a-3p exert its PCa-suppressive effects? [score:3]
These results indicate that miR-199a-3p also manifests inhibitory effects in primary PCa cells. [score:3]
Interestingly, exogenous miR-199a-3p did not significantly suppress the endogenous c-MYC protein levels in DU145 cells (Figure 7A), suggesting that c-MYC may not be the primary mediator of the miR-199a-3p effects in DU145 cells. [score:3]
Interestingly, miR-199a-3p is one of the miRNAs most dramatically underexpressed in the CD44 [+] PCa cell populations uncovered in our miRNA library screening [15]. [score:3]
Our previous study suggested that miR-199a-3p is underexpresssed in several PCa stem/progenitor cell populations, especially in CD44 [+] PCa cells [9, 15]. [score:3]
Figure 1 A. Relative expression levels of miR-199a-3p. [score:3]
miR-199a-3p suppresses clonogenic and sphere-forming properties in PCa cells. [score:3]
In both cases, we observed, in miR-199a-3p overexpressing tumors, reduced cellularity (Figure 4F-4G; compare panels a vs. [score:3]
To further investigate the tumor -inhibitory effects of miR-199a-3p, we constructed a lentiviral expression vector that encodes human miR-199A1 (Figure 4C; Supplementary Figure 1A). [score:3]
Cells were plated in six-well plate at indicated cell numbers and spheres scored on day 6. F. WB of cyclin D1 and EGFR in PC3 cells expressing miR-199a-3p (15 nM or 30 nM; 72 h). [score:3]
miR-199a-3p showed similar inhibitory effects in PC3 and LACP9 cells (Figure 2E-2G). [score:3]
As shown in Figure 4A, at 100,000 cell injections, miR-199a-3p significantly inhibited tumor growth as manifested by reduced tumor sizes. [score:3]
Nevertheless, miR-199a-3p overexpression still reduced tumor incidence and weight in LAPC9 cells (Figure 4E, b). [score:3]
Dox addition induced RFP reporter expression and increased miR-199a-3p levels (Figure 5B). [score:3]
miR-199a-3p inhibits xenograft tumor regeneration. [score:3]
Evidence that miR-199a-3p also targets c-MYC, cyclin D1, and EGFR in PCa cells. [score:3]
A. Relative expression levels of miR-199a-3p. [score:3]
miR-199a-3p was initially uncovered from our miRNA library screening for miRNAs differentially expressed in tumorigenic PCa cell subpopulations [9, 15]. [score:3]
In PCa, miR-199a-3p expression is found to be negatively associated with tumor staging and differentiation [17]. [score:3]
These results suggest that in 3 PCa cell types, miR-199a-3p overexpression causes G1 cell-cycle arrest with concomitant decrease in S or G2/M phase cells. [score:3]
miR-199a-3p overexpression significantly reduced colony formation of the CD44 [+] HPCa219 cells (Figure 3F, right). [score:3]
Overexpression of miR-199a-3p has also been reported to result in caspase -dependent and -independent apoptosis in lung cancer [21] and G1 phase cell-cycle arrest in osteosarcoma cells [22]. [score:3]
miR-199a-3p demonstrates inhibitory effects in primary human PCa (HPCa) cells. [score:3]
The miR-199a-3p expression level is generally decreased in cancers in comparison to their normal counterparts [18, 20, 22, 26]. [score:3]
We then compared the relative expression levels of miR-199a-3p (and/or miR-199a-5p) in different experimental groups (e. g., CD44 [+] vs. [score:2]
Bulk DU145 cells were also dramatically suppressed by miR-199a-3p in all of the abovementioned three assays (Figure 2B-2D). [score:2]
MiR-199a-3p is an under-studied miRNA, especially in PCa, with only one report so far showing miR-199a-3p underexpression in PCa compared to benign tissues [17]. [score:2]
Further luciferase reporter assays confirmed that miR-199a-3p partially targeted c-MYC in PC3 cells (Figure 7B). [score:2]
Figure 7 A. Western blotting (WB) showing the protein levels of c-MYC in LAPC9, PC3 and DU145 cells transfected with NC or miR-199a-3p (lanes 1-4 and 7-8) or co -transfected with pCDH-MYC vector (lanes 5-6 and 9-10) for 72 h. B. Luciferase assays showing the activity of WT or mutant MYC 3′UTR in PC3 cells expressing miR-199a-3p. [score:2]
Collectively, these observations demonstrate that miR-199a-3p negatively regulate PCSC properties. [score:2]
To determine whether miR-199a-3p possesses tumor -inhibitory effects in PCa, we carried out limiting-dilution assays (LDAs) in immunocompromised mice by monitoring tumor latency, incidence and endpoint weight. [score:2]
A. Western blotting (WB) showing the protein levels of c-MYC in LAPC9, PC3 and DU145 cells transfected with NC or miR-199a-3p (lanes 1-4 and 7-8) or co -transfected with pCDH-MYC vector (lanes 5-6 and 9-10) for 72 h. B. Luciferase assays showing the activity of WT or mutant MYC 3′UTR in PC3 cells expressing miR-199a-3p. [score:2]
As presented in Figure 5C (right), Dox induction in the lenti-199a group slowed down tumor growth (for unknown reasons, the lenti-199a group of tumors in the absence of Dox, without leakage of miR-199a-3p expression (data not shown)), also showed slightly slower growth compared to the corresponding lenti-Ctrl group). [score:2]
Collectively, these results suggest that c-MYC is regulated by miR-199a-3p in certain PCa cells. [score:2]
In this study, we present evidence for tumor suppressive functions of miR-199a-3p in both purified CD44 [+] and bulk PCa cells based on in vitro clonogenic and in vivo tumor regeneration assays as well as therapeutic experiments. [score:2]
G. Luciferase assays showing the activity of WT or mutant cyclin D1 or EGFR 3-UTR in PC3 cells expressing miR-199a-3p. [score:2]
Notably, mutation in the 3′-UTR of the miR-199a-3p binding site also restored luciferase activities to levels higher than in cells transfected with the WT 3-‘-UTR construct (Figure 7B). [score:2]
We transfected miR-199a-3p mimics or negative control (NC) oligos into either purified CD44 [+] (Figure 1B-1C) or bulk (Figure 1D-1F) PCa cells. [score:1]
miR-199a-3p reduced the live cell numbers in both purified CD44 [+] (Figure 1B-1C) and bulk (Figure 1D-1F) PCa cells. [score:1]
Preliminary studies have also revealed therapeutic efficacy of miR-199a-3p in retarding the growth of established xenograft tumors. [score:1]
miR-199a-3p exhibits therapeutic potential in a PCa xenograft mo dels. [score:1]
B. Predicted duplex formed between miR-199a-3p and 3′-UTR of CD44 by the RNA22 program. [score:1]
For example, in PC3 cells, the G1-phase cells increased from ~60% in the NC group to ~67% in the miR-199a-3p group (Figure 1G; Supplementary Figure 1D). [score:1]
Impressively, in two independent experiments, miR-199a-3p nearly completely abolished tumor regeneration from bulk DU145 cells (Figure 4B). [score:1]
First of all, we transfected miR-199a-3p and NC oligos into freshly purified CD44 [+] DU145 cells and subcutaneously implanted them into NOD/SCID mice. [score:1]
Altogether, results presented herein provide a rational for developing miR-199a-3p into anti-PCa replacement therapeutics. [score:1]
miR-199a-3p, lenti-Ctrl vs. [score:1]
NC or miR-199a-3p transfected cells (numbers indicated) were plated in 6-well plates and holoclones enumerated on day 12 (for A) and 14 (for B), respectively. [score:1]
However, studies on in vivo functions of miR-199a-3p in human cancers are generally very limited. [score:1]
Tumor pieces were quickly processed and epithelial HPCa cells were purified out (see Materials & Methods) and transfected with miR-199a-3p or NC oligos. [score:1]
Indeed, exogenously introduced miR-199a-3p reduced the CD44 protein levels in both PC3 and DU145 PCa cells (Figure 6E). [score:1]
CD44 [+] DU145 (B) and PC3 (C) cells, or bulk DU145 (D), PC3 (E), and VCaP cells (F) were transfected with 30 nM of NC or miR-199a-3p oligos and plated (20,000 cells/well) at day 0 and live cells counted at indicated days under microscope. [score:1]
pGIPZ-199A infection of LAPC9 cells for a short period of time (i. e., 48 h) led to only ~100 fold increase in miR-199a-3p levels (Figure 4E, a, right), much lower than in puromycin-selected DU145 cells (Figure 4D, a, right). [score:1]
The y-axis represents the miR-199a-3p levels in CD44 [+] cell population relative to its levels in CD44 [−] population. [score:1]
F-G. HE and IHC staining for tumors generated in NC or miR-199a-3p transfected CD44 [+] DU145 (F) and pGIPZ-Ctrl or pGIPZ-199A transduced LAPC9 (G) cells. [score:1]
400 TROP2 [+]CD44 [+] cells (purity 96.7%, below) were plated in triplicate and holoclones scored on day 9. Cells transfected with NC or miR-199a-3p (3p) oligos at 30 nM were used in above experiments (n=2-3 for each experiment). [score:1]
Importantly, CD44 protein levels were also reduced in the endpoint tumors derived from CD44 [+]DU145 cells transfected with miR-199a-3p oligos (Figure 6F), LAPC9 cells infected with the pGIPZ-199A (Figure 6G), and DU145 cells infected with the Dox-inducible lenti-199A (Figure 6H). [score:1]
In general, bulk or freshly purified CD44 [+] PCa cells and HPCa cells were transfected with NC miRNA or miR-199a-3p mimics (3p) using lipofectamine RNAiMAX (Invitrogen, Life Technology). [score:1]
Similar to c-MYC, miR-199a-3p also reduced the protein levels of cyclin D1 and EGFR in PC3 cells (Figure 7F). [score:1]
In the present study, we started by re-evaluating miR-199a-3p expression in the CD44 [+] cell population, freshly purified from DU145 cultures and two xenografts, i. e., LAPC9 and VCaP. [score:1]
Consistent with our earlier observations (Supplementary Figure 1C), transduction of DU145 cells with miR-199A1 did not cause appreciable cell death but led to significantly increased amount of miR-199a-3p (Figure 4D, a). [score:1]
We performed site-specific mutagenesis by mutating several nucleotides at the miR-199a-3p binding site on CD44 3′-UTR (Figure 6B). [score:1]
In the forgoing sections, we set out to determine the biological functions of miR-199a-3p in two AR [+]/PSA [+] (i. e., LAPC9 and VCaP) and three AR [−]/PSA [−] PCa cell line (DU145, PC3, and PPC-1) and xenograft (LAPC9 and VCaP) mo dels. [score:1]
These results, collectively, reveal a therapeutic potential of miR-199a-3p in PCa. [score:1]
When we transfected miR-199a-3p oligos into LAPC9 and PC3 cells, endogenous c-MYC protein levels decreased (Figure 7A; lanes 2 and 4 vs. [score:1]
In contrast, both DU145 and LAPC9 tumors showed very little apoptotic (i. e., lamin A [+]) cells and there were no differences between control and miR-199a-3p tumors (Figure 4F-4G; compare panels e vs f). [score:1]
The relative expression levels of miR-199a-3p and miR-199a-5p were measured by RT-qPCR. [score:1]
H. mTOR, phosphorylated AKT and AKT were determined by WB in DU145 cells treated with NC or miR-199a-3p (10 nM, 72 h). [score:1]
Neither miR-199a-3p nor NC induced appreciable cell senescence in the 3 PCa cell types (date not shown). [score:1]
Indeed, we identified a potential miR-199a-3p binding site in the c-MYC CDS (Supplementary Figure 3A). [score:1]
miR-199a-3p exhibits therapeutic potential in PCa cells. [score:1]
G-H. DNA content analysis in bulk PC3 (G) or DU145 (H) cells transfected with miR-199a-3p or NC (30 nM, 48 h). [score:1]
Consequently, we studied the biological functions of miR-199a-3p in 4 HPCa specimens with ~100% tumor involvement (Supplementary Figure 2A-2D). [score:1]
In silico analysis identified a putative miR-199a-3p binding site at the 3′-UTR of CCND1 and EGFR, respectively (Supplementary Figure 3A). [score:1]
For therapeutic experiments, DU145 cells were infected with negative control (lenti-Ctrl) and miR-199a-3p lentivirus (lenti-199A) and subcutaneously implanted into NOD/SCID female mice. [score:1]
Interestingly, accompanying the increase in G1-phase cells, miR-199a-3p reduced S-phase cells in PC3 (Figure 1G; Supplementary Figure 1D) but reduced G2/M-phase cells in DU145 (Figure 1H; Supplementary Figure 1D) and PPC-1 (Supplementary Figure 1B; Supplementary Figure 1D) cells. [score:1]
D-E. mRNA (D) and protein (E) of CD44 in NC or miR-199a-3p transfected DU145 and PC3 cells. [score:1]
In contrast, no significant difference was observed between NC and miR-199a-3p treated PCa cells in early apoptotic, late apoptotic or late necrotic cells (Supplementary Figure 1E). [score:1]
We observed that miR-199a-3p treatment increased the % of G1-phase cells in PC3, DU145, and PPC-1 cultures (Figure 1G and 1H; Supplementary Figure 1B-1D). [score:1]
Cells were transfected with NC or miR-199a-3p oligos at 30 nM. [score:1]
B. qPCR analysis of miR-199a-3p after Dox treatment for 72 h (left panel). [score:1]
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[+] score: 327
To explore whether upregulated P53 expression was attributed to affected miR199a-3p expression in cardiac c-kit [+] cells, we constructed P53 lentivirus to infect cardiac c-kit [+] cells, and then detected that overexpression of P53 increased miR199a-3p levels. [score:10]
Taken together, our data confirmed one new mechanism by which the downregulation of miR199a-3p miRNAs and the responsive upregulation of its target protein CABLES1 induced the P53 increase. [score:9]
miR199a-3p regulates cardiac c-kit [+] cell proliferation and apoptosis through targeting CABLES1To determine whether the regulation of proliferation and apoptosis of cardiac c-kit [+] cells through miR199a-3p is directly mediated by CABLES1, we performed rescue experiments by transfecting the miR199a-3p and CABLES1 lentiviral vectors into cardiac c-kit [+] cells and examining CABLES1 expression levels through (Fig.   5a). [score:8]
Here, we hypothesized that the miR199a expression and activity in human failing myocardium may be a result of upregulation of P53 expression, and results in the survival of cardiac c-kit [+] cells. [score:8]
In cardiomyocytes, miR199a downregulates hypoxia-inducible factor 1 and the stabilization of the proapoptotic factor p53 [9], and is one of the factors regulating cell size; its overexpression in cardiomyocytes leads to hypertrophy [41]. [score:7]
In addition, rescue experiments further confirmed that miR199a-3p functioned through downregulating CABLES1 expression directly to promote cell proliferation and reduce cell apoptosis. [score:7]
Meanwhile, we identified miR199a-3p as a proliferation- and apoptosis -associated regulator impacted through Cdk5 and Abl enzyme substrate 1 (CABLES1) targeting, and also attributed their repression to P53 protein expression. [score:6]
Meanwhile, the upregulation of p53 may be critical in the modulation of heart failure [20, 21], and has also been shown to activate the miR199a-3p expression at the post-transcriptional level in induced pluripotent stem cells (iPSCs) [22]. [score:6]
Increased expression of miR199a-3p may be proapoptotic by virtue of its ability to downregulate the survival kinase ERK2 [36]. [score:6]
We suppose that upregulation of p53 may the initial factor, and p53 elevated the miR199a-3p expression. [score:6]
Expression of miR199a is downregulated in heart failure patients. [score:6]
The high expression of P53 ultimately offset the miR199a-3p upregulation, which provided evidence of a novel negative feedback loop that likely contributes to cardiac c-kit [+] cell proliferation and apoptosis (Fig.   9). [score:6]
To determine whether miR199a-3p directly regulates CABLES1, we performed luciferase reporter experiments and observed that the luciferase activity of CABLES1-WT was markedly reduced after transfection with the miR199a-3p lentiviral vectors for 24 h. However, single mutations completely abolished the repression induced by miR199a-3p (Fig.   4b), indicating that miR199a-3p could specifically target the binding sites in the 3’ UTR of CABLES1. [score:6]
Herein, we showed that the function of miR199a-3p to promote cell proliferation and inhibit cell apoptosis was also attributed to its repression of P53 protein expression. [score:5]
Moreover, overexpression of miR199a in cardiomyocytes results in disruption of sarcomere structure and, conversely, suppression of miR199a abolishes this phenotype [15]. [score:5]
The putative target sites of miR199a-3p were predicted using TargetScan, microRNA and PicTar software. [score:5]
Overexpression of miR199a-3p was found to inhibit the invasiveness of tumor cells [36] and to modulate their sensitivity to doxorubicin -induced apoptosis in hepatocellular carcinoma [37]. [score:5]
The correlation between miR199a-3p expression and the protein levels of the target genes was examined through Pearson’s correlation analysis. [score:5]
To determine whether the regulation of proliferation and apoptosis of cardiac c-kit [+] cells through miR199a-3p is directly mediated by CABLES1, we performed rescue experiments by transfecting the miR199a-3p and CABLES1 lentiviral vectors into cardiac c-kit [+] cells and examining CABLES1 expression levels through (Fig.   5a). [score:5]
Analysis of total RNA to determine the relative expression of miR199a-3p targets through qRT-PCR in cardiac c-kit [+] cells at 48 h after transfection. [score:5]
miR-199a-3p has been found to be highly expressed in ovarian and breast cancer [33], but has low expression in hepatocellular carcinoma [34] and in bladder cancer [35]. [score:5]
For miR199a-3p upregulation or knockdown, a miR199a-3p Lentivirus and anti-miR Lentivirus were purchased from GenePharma (Shanghai, China). [score:5]
These data demonstrated that miR199a-3p could promote cell proliferation and inhibit cell apoptosis through regulating P53. [score:4]
After transfection, knockdown of P53 decreased miR199a-3p expression (Fig.   8d). [score:4]
Together, these results indicated that CABLES1 is a direct target of miR199a-3p in cardiac c-kit+ cells. [score:4]
The upregulation of miR199a-3p can promote cell proliferation and protects against angiotensin II -induced apoptosis. [score:4]
miR199a-3p regulates cardiac c-kit [+] cell proliferation and apoptosis through targeting CABLES1. [score:4]
Finally, the increase in miR199-3p may ultimately offset the P53 upregulation in heart failure. [score:4]
c Regulation of endogenous expression of CABLES1 by treatment with the miR199a-3p lentiviral vectors and anti-miR199a-3p. [score:4]
CABLES1 Cdk5 and Abl enzyme substrate 1, Con control (* p < 0.05, compared to miR-con) We examined mRNA expression for each target in response to miR199a-3p and anti-miR199a-3p. [score:4]
miR199a-3p is involved in regulation of cardiac c-kit [+] cell proliferation and apoptosisTo examine whether miR199a-3p affects cell proliferation and apoptosis in cardiac c-kit [+] cells we first tested the miR199a-3p expression after 200 nM angiotensin II treatment for 48 h in cardiac c-kit [+] cells. [score:4]
CABLES1 Cdk5 and Abl enzyme substrate 1, Con control (* p < 0.05, compared to miR-con) We examined mRNA expression for each target in response to miR199a-3p and anti-miR199a-3p. [score:4]
To address the mechanism through which miR199a-3p regulates cardiac c-kit [+] cell survival, we examined the predicted targets of miR199a-3p. [score:4]
Area under the curve (A) sensitivity and specificity were 0.98 and 0.58, respectively ROC curve analysis suggested that the expression levels of miR199a-3p could be used as a strong diagnostic predictor of heart failure, with an area under the curve (AUC) of 0.98 (Fig.   1b). [score:3]
These data confirmed that the effects of miR199a-3p on proliferation and apoptosis in cardiac c-kit [+] cells could be attributed to the target protein CABLES1. [score:3]
Research has suggested that miR199 may have significant differential expression in the myocardium during heart failure. [score:3]
a Analysis of total RNA to determine the relative expression of miR199a-3p through qRT-PCR in cardiac c-kit [+] cells at 48 h after treatment with 200 nM angiotensin II (AngII). [score:3]
Computational screening showed that one of the potential target sites of miR199a-3p was in the 3’ UTR of mouse and human CABLES1. [score:3]
However, no significant change in the expression of miR199a-5p was detected. [score:3]
The results suggested that cardiac c-kit [+] cells express lower proliferation (3.2 ± 0.4% and 4.2 ± 0.3%, respectively) in combination transfection of miR199a-3p and CABLES1 than the control group (6.5 ± 0.6% and 10.5 ± 0.7%, respectively) (Fig.   5c). [score:3]
a Expression of miR199a-3p and miR199a-5p in the plasma from 60 heart failure patients and 60 healthy donors was detected by RT-PCR. [score:3]
The functional activity of miR199a-3p was assessed in cardiac c-kit [+] cells after forced expression of this miRNA or its corresponding anti-miRNA using lentiviral vectors. [score:3]
We performed rescue experiments by transfection with the combination of anti-miR199-3p and shRNA-CABLES1 in cardiac c-kit [+] cells and detected the protein expression by (Fig.   6a). [score:3]
Fig. 4CABLES1 is a target of miR199a-3p in cardiac c-kit [+] cells. [score:3]
Collectively, our findings uncover one new mechanism by which P53 induced miR199a-3p expression and, in turn, miR199-3p decreased P53 activity. [score:3]
A negative feedback loop comprising miR199a-3p, CABLES1, and P53 is involved in cardiac c-kit [+] cellsIt had been reported that P53 activates miR199a-3p expression at the post-transcriptional level [22]. [score:3]
We performed RT-PCR in plasma samples from 60 heart failure patients and 60 healthy donors to analyze the expression of miR199a-3p and miR199a-5p. [score:3]
The role of miR199a-3p as a tumor suppressor or oncogene has been studied by several groups [30– 32]. [score:3]
We further demonstrated that P53 induced miR199a-3p expression and, in turn, miR199-3p decreased P53 activity. [score:3]
We performed experiments by transfection with the combination of anti-miR199-3p and shRNA-CABLES1 in cardiac c-kit [+] cells and detected the P53 protein expression by (Fig.   6a). [score:3]
Area under the curve (A) sensitivity and specificity were 0.98 and 0.58, respectively ROC curve analysis suggested that the expression levels of miR199a-3p could be used as a strong diagnostic predictor of heart failure, with an area under the curve (AUC) of 0.98 (Fig.   1b). [score:3]
To examine whether miR199a-3p affects cell proliferation and apoptosis in cardiac c-kit [+] cells we first tested the miR199a-3p expression after 200 nM angiotensin II treatment for 48 h in cardiac c-kit [+] cells. [score:3]
Therefore, these data showed that miR199a-3p promotes cell proliferation and inhibits apoptosis in cardiac c-kit [+] cells. [score:3]
To identify the potential targets of miR199a-3p that would facilitate cell growth, we used bioinformatic search tools. [score:3]
CABLES1 is a target for miR199a-3p. [score:3]
It had been reported that P53 activates miR199a-3p expression at the post-transcriptional level [22]. [score:3]
Indeed, overexpression of P53 increased miR199a-3p levels by approximately 3.8-fold (Fig.   8b). [score:3]
Fig. 1Significantly decreased miR199a-3p expression was detected in heart failure patients. [score:3]
Furthermore, the expression of miR590 or miR199a in the heart after infarction exerts a marked beneficial effect in reducing infarct size and in improving cardiac function [16]. [score:3]
We examined the protein expression levels of CABLES1 in response to miR199a-3p. [score:3]
To identify the mechanisms associated with miR199a-3p and P53 in cardiac c-kit [+] cells, we examined P53 expression in response to miR199a-3p and CABLES1. [score:3]
b ROC curve analysis of miR199a-3p and miR199a-5p expression in patients. [score:3]
Furthermore, it was reported that miR199a-3p targeting of CABLES1 might play an important role in breast cancer tumor progression [38] and human osteosarcoma [31], as well as supporting its role as an oncomiR in a pre-leukemic mouse mo del [39]. [score:3]
We found that expression of miR199a-3p promoted proliferation and survival of cardiac c-kit [+] cells. [score:3]
We found that miR199a-3p had lower expression in cardiac c-kit [+] cells after apoptosis (Fig.   2a). [score:3]
b Expression of miR199a-3p mRNA in cardiac c-kit [+] cells transfected with the P53 lentivirus. [score:3]
d Expression of miR199a-3p mRNA in cardiac c-kit [+] cells transfected with the shRNA-p53. [score:3]
miR199a-3p is involved in regulation of cardiac c-kit [+] cell proliferation and apoptosis. [score:2]
The EdU results showed that overexpression of miR199a-3p was able to promote cell proliferation (6.1 ± 0.28%) compared with the control group (2.4 ± 0.25%), whereas the anti-miR199a-3p group (1.7% ± 0.33%) exhibited lower proliferation (Fig.   2b). [score:2]
The miR199 family plays an important role in hypoxia -induced cell death through regulation of hypoxia-inducible factor-1a (HIF-1a) and the stabilization of the proapoptotic factor p53 [9]. [score:2]
The regulation of miR199a-3p on cell proliferation and apoptosis is involved in P53. [score:2]
Previous study showed that miR199a-3p has been found to be dysregulated in end-stage heart failure [40]. [score:2]
These assays demonstrated that P53 induced miR199a-3p expression and, in turn, miR199-3p decreased P53 activity. [score:2]
The role of miR199a has been described in STAT-3 knockout mice which develop spontaneous heart failure [15]. [score:2]
We demonstrated a significantly decreased expression of miR199a-3p in heart failure samples compared with healthy donors. [score:2]
Fig. 9Schematic illustration of the feedforward/positive feedback loop between miR199a-3p, CABLES1, and P53. [score:1]
c Cell cycle distribution of cardiac c-kit [+] cells after transfection with the miR199a-3p and CABLES1 lentiviral vectors. [score:1]
A negative feedback loop comprising miR199a-3p, CABLES1, and P53 is involved in cardiac c-kit [+] cells. [score:1]
a The nucleotide sequence of miR199a-3p, the predicted binding sites of miR199a-3p, and the mutated nucleotides (underlined) in the 3′ UTR of CABLES1. [score:1]
Sequences were: mature miR199a-3p, 5′-ACAGUAGUCUGCACAUUGGUUA-3′; and anti miR199a-3p, 5′-AACCAAUGU-GCAGACUACUGUA-3′. [score:1]
d Western blot analysis of proteins from cardiac c-kit [+] cells transfected with the miR199a-3p lentiviral vectors and anti-miR199a-3p. [score:1]
Interestingly, miR199a-3p was significantly decreased in the plasma of heart failure patients. [score:1]
The miR199a-3p binding site in the seed sequences within the 3’ UTR of CABLES1 mRNA is illustrated in Fig.   4a. [score:1]
CABLES1 Cdk5 and Abl enzyme substrate 1, Con control, PI propidium iodide (* p < 0.05) The EdU results show that the CABLES1 transfection mitigated the cell amplification (1.8 ± 0.31% and 2.1 ± 0.34%, respectively) which results from miR199a-3p treatment or negative control (2.6 ± 0.21% and 6.6 ± 0.28%, respectively), and results in reduced cell proliferation (Fig.   5b). [score:1]
Lentiviral vectors of miR199a-3p, its corresponding anti-miRNA, or short hairpin RNA against Cables1 were transfected into cells. [score:1]
Fig. 8A negative feedback loop comprising miR199a-3p, CABLES1, and P53 is involved in cardiac c-kit [+] cells. [score:1]
Furthermore, the apoptosis by angiotensin II was much more increased in the combination group (37.7 ± 1.9%) or the CABLES1 transfection group (38.6 ± 2.1%) than the miR199a-3p group (10.8 ± 1.3%) or the negative control (25.7 ± 2.3%) (Fig.   5d). [score:1]
a Western blot analysis of proteins from cardiac c-kit [+] cells transfected with the anti-miR199a-3p and shRNA-CABLES1 lentiviral vectors (* p < 0.05). [score:1]
c Cell cycle distribution of cardiac c-kit [+] cells after transfection with the anti-miR199a-3p and shRNA-CABLES1 lentiviral vectors. [score:1]
Therefore, miR199a-3p and P53 are coupled through CABLES1 and comprise a negative feedback loop. [score:1]
b The cells were stained with EdU and DAPI after transfection with the miR199a-3p and anti-miR199a-3p. [score:1]
The results showed that P53 activity was decreased 48% in the miR199a-3p group and increased 4.2-fold in the anti-miR199a-3p group (Fig.   7). [score:1]
We observed that CABLES1 protein levels were significantly decreased by treatment with the miR199a-3p lentiviral vectors and increased by treatment with the anti-miR199a-3p (Fig.   3d). [score:1]
The WT Wnt TOPflash reporter was transfected into cardiac c-kit [+] cells together with the mimic-control (con), miR199a-3p, and anti-miR199a-3p. [score:1]
CABLES1 Cdk5 and Abl enzyme substrate 1, Con control, PI propidium iodide (* p < 0.05) The EdU results show that the CABLES1 transfection mitigated the cell amplification (1.8 ± 0.31% and 2.1 ± 0.34%, respectively) which results from miR199a-3p treatment or negative control (2.6 ± 0.21% and 6.6 ± 0.28%, respectively), and results in reduced cell proliferation (Fig.   5b). [score:1]
Fig. 7The regulation of miR199a-3p on cell proliferation and apoptosis is involved in P53 (* p < 0.05, compared to miR-con). [score:1]
For cell cycle analysis, the cells were harvested at 48 h after transfection with the miR199a-3p or anti miR199a-3p, then washed twice with PBS and fixed in 75% ethanol overnight. [score:1]
a Western blot analysis of proteins from cardiac c-kit [+] cells transfected with the miR199a-3p and CABLES1 lentiviral vectors (* p < 0.05). [score:1]
Therefore, miR199a-3p and P53 are coupled through CABLES1 and comprise a novel negative feedback loop that likely contributes to cardiac c-kit [+] cell proliferation and apoptosis. [score:1]
Relative luciferase (LUC) activity was measured and plotted analysis revealed that treatment with miR199a-3p decreased endogenous P53 expression. [score:1]
U6 was employed for miR199a-3p template normalization. [score:1]
a Cell cycle distribution of cardiac c-kit [+] cells after transfection with the miR199a-3p and anti-miR199a-3p for 48 h. b Representative annexin V/PI flow cytometry analysis of cardiac c-kit [+] cells. [score:1]
, Cambridge, MA, USA) was co -transfected with the miR199a-3p and anti-miR Lentivirus, along with 5 ng of a Renilla LUC reporter plasmid using Oligofectamine (Invitrogen) in cardiac c-kit [+] cells (60% confluency). [score:1]
Relative luciferase (LUC) activity was measured and plotted Western blotting analysis revealed that treatment with miR199a-3p decreased endogenous P53 expression. [score:1]
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5
[+] score: 228
Other miRNAs from this paper: mmu-mir-199a-1, mmu-mir-199b
We found that miR-199a/b-3p over -expression down-regulated the mRNA and protein expression of PAK4, while PAK4 silencing suppressed MGC-803 and SGC-7901 cell proliferation. [score:10]
MiR-199a/b-3p over -expression and PAK4 silencing inhibited cell proliferation and diminished the activation of p-MEK and p-ERK in MGC-803 and SGC-7901 cells, and miR-199a/b-3p over -expression reduced PAK4 expression. [score:9]
Because miRNAs function mainly through suppressing their target genes, we further predicted and analyzed the target genes of miR-199a/b-3p that functions in GC pathogenesis by TargetScan. [score:9]
Moreover, In our in vivo study, we also found that miR-199a/b-3p over -expression suppressed GC cell proliferation and PAK4 expression. [score:7]
miR-199a/b-3p inhibits GC cell proliferation via down -regulating PAK4/MEK/ERK signaling pathway and may be a novel prognostic biomarker and a potential therapeutic target for GC patients. [score:6]
We found that both miR-199a/b-3p over -expression and PAK4 silencing down-regulated the activation of p-MEK and p-ERK in MGC-803 and SGC-7901 cells. [score:6]
MiR-199a/b-3p over -expression suppressed MGC-803 cell growth and PAK4 expression in nude mice. [score:6]
This results indicate that miR-199a/b-3p can suppresses GC cell proliferation through down -regulating PAK4 expression in vivo. [score:6]
Therefore, we reasoned that miR-199a/b-3p inhibits GC cell proliferation may through down -regulating PAK4 expression. [score:6]
MiR-199a/b-3p down-regulates PAK4 expression in vitro. [score:5]
Recent studies showed that miR-199a/b-3p function as a tumor-suppressor gene and inhibits cell proliferation in some cancers, such as human hepatocellular carcinoma [7], papillary thyroid carcinoma [9], endometrioid adenocarcinoma [10], renal cell carcinoma [11], osteosarcoma [12], ovarian cancer [13] and breast cancer [14]. [score:5]
TargetScan prediction of miR-199a/b-3p target sites in PAK4; Table S4. [score:5]
We found that miR-199a/b-3p expression was decreased in GC tissues and cell lines, and identified it as a critical suppressor of GC cell proliferation in both in vitro and in vivo studies. [score:5]
org), PAK4 not only contains two putative conserved target sites of miR-199a/b-3p (Additional file 1: Table S3), but also is over-expressed [15] and have an oncogenic role in GC [16]. [score:5]
This results are in line with previous findings showing that miR-199a/b-3p over -expression can inhibit cell proliferation in human hepatocellular carcinoma [7] and breast cancer [14]. [score:5]
Our results show that miR-199a/b-3p expression was decreased in GC and it can inhibit GC cell proliferation. [score:5]
Interestingly, some previous studies showed that miR-199a/b-5p was highly expressed in GC tissues compared with normal adjacent tissues and that miR-199a/b-5p functions as an oncogene in GC by targeting klotho [18– 20]. [score:4]
MiR-199a/b-3p inhibits GC proliferation by decreasing PAK4 expression in vivo. [score:4]
Our results show that miR-199a/b-3p inhibits GC cell proliferation via down -regulating PAK4/MEK/ERK signaling pathway. [score:4]
MiR-199a/b-3p functions as a tumor suppressor in diverse cancers, but its expression, function, and mechanism in GC remain unclear. [score:4]
This study is first focused on miR-199a/b-3p expression and its role in regulating GC cell proliferation. [score:4]
In summary, our results show that miR-199a/b-3p inhibits GC cell proliferation via down -regulating PAK4/MEK/ERK signaling pathway. [score:4]
Top 25 predicted targets of miR-199-3p/5p sorted by aggregate P [CT] (DOCX 535 kb) ERK Extracellular signal-regulated kinase. [score:4]
Song G, Zeng H, Li J, Xiao L, He Y, Tang Y, Li Y: miR-199a regulates the tumor suppressor mitogen-activated protein kinase kinase kinase 11 in gastric cancer. [score:4]
Our aim is to explore miR-199a/b-3p expression and its role in regulating GC cell proliferation. [score:4]
MiR-199a/b-3p over -expression and PAK4 knockdown inhibited the cell proliferation ability of GC cell line 7901 in vitro as analyzed by CCK-8 assay; Table S1. [score:4]
We also showed that miR-199a/b-3p functions as a GC suppressor via down -regulating PAK4/MEK/ERK signaling pathway. [score:4]
These results indicate that miR-199a/b-3p can inhibit GC cell proliferation in a dose -dependent way. [score:3]
Moreover, our finding shows that miR-199a/b-3p functions as an anti-oncogene in GC by targeting PAK4. [score:3]
We found that the expression levels of miR-199a/b-3p were significantly lower in the cancer tissues than in the normal tissues (Fig.   1a). [score:3]
c MGC-803 and SGC-7901 cells were transfected with negative control (NC), miR-199a/b-3p mimics (50 nM), transfection reagent only (Mock) or PAK4 siRNA (50 nM), at 48 h post-transfection, protein expression of p-MEK, p-ERK and β-actin (internal control) was detected using western blotData are shown as the mean ± SD of three independent experiments. [score:3]
Although miR-199a/b-5p and miR-199a/b-3p are the two nature forms of miR-199a/b, we identified that they not only have different miRNA sequences (Additional file 1: Table S4) but also have numerous different predicted target genes (Additional file 1: Table S5) through using bioinformatic analyses. [score:3]
Their miR-199a/b-3p expression was detected using real-time PCR. [score:3]
These findings suggest that miR-199a/b-5p and miR-199a/b-3p play opposite roles in GC may by targeting different downstream genes. [score:3]
Enrichment analysis of predicted miR-199a/b-3p targets in GCBI pathway database; Figure S2. [score:3]
c MGC-803 and SGC-7901 cells were transfected with negative control (NC) or miR-199a/b-3p mimics at a final concentration of 10 or 50 nM, at 48 h post-transfection, miR-199a/b-3p expression was detected using real-time PCR. [score:3]
Real-time PCR was performed to determine miR-199a/b-3p expression in GC tissues and normal adjacent tissues as well as normal gastric mucosal cell line GES-1 and GC cell lines MGC-803 and SGC-7901. [score:3]
As the MEK/ERK pathway plays a critical role in PAK4 -induced cell proliferation [17], we subsequently explore whether the MEK/ERK pathway was involved in miR-199a/b-3p antitumor effect by targeting PAK4 in GC. [score:3]
To further confirm that miR-199a/b-3p suppresses GC cell proliferation. [score:3]
To explore it, we transfected MGC-803 and SGC-7901 cells with negative control (NC), miR-199a/b-3p mimics, transfection reagent only (Mock) or PAK4 siRNA, and assessed their protein expression by western blot. [score:3]
a, b MGC-803 and SGC-7901 cells were transfected with negative control (NC) or miR-199a/b-3p mimics at a final concentration of 10 or 50 nM, at 48 h post-transfection, PAK4 mRNA and protein expression was detected using real-time PCR and western blot, respectively. [score:3]
Together, these results show that miR-199a/b-3p inhibits GC cell proliferation by the miR-199a/b-3p/PAK4/MEK/ERK axis. [score:3]
cn) to identify signaling pathways enriched by hundreds of predicted targets of miR-199a/b-3p. [score:3]
MiR-199a/b-3p inhibits GC cell proliferation in vitro. [score:2]
In order to investigate miR-199a/b-3p expression in GC, we compared the expression levels of miR-199a/b-3p between the GC tissues and the normal adjacent tissues by real-time PCR. [score:2]
We found that the expression levels of miR-199a/b-3p were significantly lower in GC tissues compared with normal adjacent tissues. [score:2]
Similarly, miR-199a/b-3p expression was decreased in the GC cell lines MGC-803 and SGC-7901 compared with the normal gastric mucosal cell line GES-1. In addition, we also found that the viable cell levels were significantly lower in MGC-803 and SGC-7901 cells transfected with miR-199a/b-3p mimics than in cells transfected with negative control. [score:2]
MiR-199a/b-3p expression was significantly decreased in GC tissues and GC cell lines MGC-803 and SGC-7901. [score:2]
We found that MGC-803 and SGC-7901 cells transfected with miR-199a/b-3p mimics had significantly increased expression levels of miR-199a/b-3p in a dose -dependent way (Fig. 1c), while had significantly decreased viable cell levels at 48 and 72 h post-transfection as compared with cells transfected with negative control (Fig. 1d). [score:2]
To address this issues, we transfected MGC-803 and SGC-7901 cells with miR-199a/b-3p mimics or negative control, and compared their expression levels of PAK4 mRNA and protein. [score:2]
b MiR-199a/b-3p expression in a normal gastric mucosa cell line GES-1, GC cell lines MGC-803 and SGC-7901 was detected using real-time PCR. [score:2]
We found that MGC-803 and SGC-7901 cells transfected with miR-199a/b-3p mimics had significantly decreased expression levels of PAK4 mRNA and protein in a dose -dependent way as compared with cells transfected with negative control (Fig.   2a and b). [score:2]
To our knowledge, this is the first study to demonstration that the miR-199a/b-3p/PAK4/MEK/ERK axis is involved in regulating GC cell proliferation. [score:2]
In addition, Compared with the normal gastric mucosal cell line GES-1, the GC cell lines MGC-803 and SGC-7901 had lower expression levels of miR-199a/b-3p (Fig. 1b). [score:2]
The purpose of this study is to investigate miR-199a/b-3p expression and its role in regulating GC cell proliferation. [score:2]
MiR-199a/b-3p suppresses PAK4/MEK/ERK pathway in GC in vitro. [score:2]
We transfected MGC-803 cells with negative control (NC) or miR-199a/b-3p mimics, then implanted these cells into nude mice subcutaneously, finally compared the size of their xenograft tumors and the expression of PAK4 mRNA and protein. [score:2]
Therefore, we investigate whether the MEK/ERK pathway was involved in miR-199a/b-3p antitumor effect by targeting PAK4 in GC. [score:1]
a MGC-803 cells transfected with negative control (NC) or 50 nM miR-199a/b-3p mimics respectively, were implanted into the axillary fossae of each mouse, after 2 weeks, xenograft tumors were removed intactly. [score:1]
Its two putative hairpin precursors in human map to chromosome 19 and chromosome 1, and the mature forms of its excised miR sequences are named miR-199a/b-5p and miR-199a/b-3p [7, 8]. [score:1]
To assess the anti-proliferation capacity of miR-199a/b-3p in vivo, 2 × 10 [6] MGC-803 cells transfected with negative control (NC) or 50 nM miR-199a/b-3p mimics respectively, were suspended in 0.2 ml sterile saline and subsequently implanted subcutaneously into the axillary fossae of each mouse. [score:1]
The association of miR-199a/b-3p relative expression with the clinicopathological characteristics in 20 GC patients are showed in Additional file 1: Table S1. [score:1]
Sequences of miR-199a/b-5p and miR-199a/b-3p; Table S5. [score:1]
GC xenograft nude mice were used to determine miR-199a/b-3p function in cell proliferation. [score:1]
The association of miR-199a/b-3p relative expression with the clinic-pathological characteristics in 20 GC patients; Table S2. [score:1]
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[+] score: 172
MSCs+miR-199a-5p, MSCs transfected with miR-199a-5p mimics; MSCs+miR-199a-5p NC, MSCs transfected with miR-199a-5p mimics negative control; MSCs+miR-199a-5p inhibitor, MSCs transfected with miR-199a-5p inhibitor; MSCs+miR-199a-5p inhibitor NC, MSCs transfected with miR-199a-5p inhibitor negative control. [score:9]
It could directly upregulate VEGFA expression by inhibiting miR-199a-5p as a competing endogenous RNA. [score:9]
Enforced expression of miR-199a-5p provokes downregulated expression of proangiogenic factors in hypoxic multiple myeloma cells and impairs the migration of ECs and angiogenesis [34]. [score:8]
H/SD: hypoxia/serum deprivation, MSCs: mesenchymal stem cells, NC: scramble negative control We further examined the expression of lncRNA-H19 and miR-199a-5p in the condition of lncRNA-H19 overexpression or knockdown under both normoxia and H/SD conditions in order to identify whether miR-199a-5p could be regulated by lncRNA-H19. [score:7]
pc3.1-H19, pc3.1-H19 scramble negative control (H19 NC), H19 siRNA(si-H19), scramble negative control of H19 siRNA (si-H19 NC), miR-199a-5p mimics, miR-199a-5p mimics negative control (miR-199a-5p NC), miR-199a-5p inhibitor and miR-199a-5p inhibitor negative control (miR-199a-5p inhibitor NC) were transiently transfected into MSCs. [score:7]
H/SD: hypoxia/serum deprivation, MSCs: mesenchymal stem cells, NC: scramble negative controlWe further examined the expression of lncRNA-H19 and miR-199a-5p in the condition of lncRNA-H19 overexpression or knockdown under both normoxia and H/SD conditions in order to identify whether miR-199a-5p could be regulated by lncRNA-H19. [score:7]
The aforementioned luciferase reporter assay and the subsequent functional detection confirmed that lncRNA-H19 could competitively inhibit miR-199a-5p to further upregulate the expression of VEGFA. [score:7]
To determine whether VEGFA was negatively regulated by miR-199a-5p, its expression was examined in MSCs treated with miR-199a-5p mimics or miR-199a-5p inhibitor. [score:6]
MicroRNA-199a-5p (miR-199a-5p) targeted and downregulated vascular endothelial growth factor A (VEGFA). [score:6]
Here, we also found that the expression level of VEGFA was significantly receded after the transfection of miR-199a-5p mimics, whereas miR-199a-5p inhibition remarkably raised its level, suggesting that VEGFA was negatively regulated by miR-199a-5p. [score:6]
In the present work, we demonstrated that lncRNA-H19 enhanced MSCs survival and angiogenic capacity by acting as a molecular sponge to competitively inhibit miR-199a-5p and further modulated its target VEGFA. [score:5]
However, its inhibition elevated the expression of miR-199a-5p. [score:5]
Downregulation of miR-199a-5p in pulmonary microvascular ECs accelerates proliferation and angiogenesis [33]. [score:4]
In summary, we demonstrated the role of lncRNA-H19 in enhancing MSCs survival and angiogenic capacity in vitro, and identified a potential ceRNA network by which lncRNA-H19 acted as a molecular sponge for miR-199a-5p to regulate the expression of VEGFA in MSCs. [score:4]
LncRNA-H19 targeted and negatively regulated miR-199a-5p. [score:4]
Fig. 7LncRNA-H19 targeted and negatively regulated miR-199a 5p. [score:4]
LncRNA-H19 transfection led to a decreased level of miR-199a-5p, accompanied with an elevated expression of VEGFA. [score:3]
H/SD: hypoxia/serum deprivation, MSCs: mesenchymal stem cells, NC: scramble negative control VEGFA has been validated as a target of miR-199a-5p and their interaction has been predicted by bioinformatics and confirmed by functional experiments in MSCs in previous studies (Fig.   6a) [20]. [score:3]
Further displayed that miR-199a-5p was inversely correlated with lncRNA-H19 expression. [score:3]
b Expression of miR-199a-5p detected by qRT-PCR. [score:3]
Mutant H19 contained a mutation site eliminating targeting by miR-199a-5p, and its primer sequences were as follows: forward, 5′-GCGGAAAGGGCCCACAGTGGACTTGAGCTCTGATATGCCCTAACCGCTCAGTCCCTGG-3′; and reverse, 5′-CCAGGGACTGAGCGGTTAGGGCATATCAGAGCTCAAGTCCACTGTGGGCCCTTTCCGC-3′. [score:3]
It was shown that the protein and mRNA levels of VEGFA were obviously decreased in the MSCs+miR-199a-5p group under both normoxia and H/SD conditions (P < 0.01, Fig.   6c, d; P < 0.01, Fig.   6e), while the MSCs+miR-199a-5p inhibitor group presented a distinct augmentation of VEGFA (P < 0.01, Fig.   6c, d; P < 0.01, Fig.   6e). [score:3]
We explored the potential role of lncRNA-H19 as a ceRNA to inhibit miR-199a-5p. [score:3]
These results reflected that lncRNA-H19 functioned as a ceRNA and modulated the expression of miR-199a-5p to improve MSCs survival and angiogenic capacity. [score:3]
qRT-PCR were performed to explore expression of miR-199a-5p in different experimental groups under normoxia and H/SD conditions. [score:3]
In the present work, to fully understand the functionary mechanism of lncRNA-H19, we speculated that it acted as a sponge for miR-199a-5p to mediate the target gene VEGFA. [score:3]
We showed that miR-199a-5p was a target of lncRNA-H19. [score:3]
LncRNA-H19 overexpression resulted in a significant decline in the level of miR-199a-5p under both normoxia and H/SD conditions. [score:3]
a Potential consequential paring of VEGFA -targeted region and miR-199a-5p. [score:3]
The miR-199a-5p expression status was detected by the (P < 0.01; Fig.   6b). [score:3]
VEGFA has been certified as a target of miR-199a-5p [20]. [score:3]
The MSCs+H19 group presented an obvious downregulation of miR-199a-5p compared with the MSCs+H19 NC and MSCs groups (P < 0.01, Fig.   7c). [score:3]
However, both miR-199a-5p and VEGFA presented inverse alterations in the condition of lncRNA-H19 knockdown. [score:2]
All these results demonstrated that miR-199a-5p was a negative regulator of VEGFA. [score:2]
Dual luciferase report and qRT-PCR assays confirmed that lncRNA-H19 could target miR-199a-5p. [score:2]
MiR-199a-5p was a target of lncRNA-H19. [score:2]
Long noncoding RNA H19 Vascular endothelial growth factor A Mesenchymal stem cells MiR-199a-5p Survival Angiogenic capacity Stem cell transplantation has emerged as a novel therapeutic approach for the treatment of cardiovascular disease [1]. [score:2]
LncRNA-H19 and miR-199a-5p exhibit opposite regulatory effects in a plethora of biological activities of organisms [41– 44]. [score:2]
c Alterations of miR-199a-5p after lncRNA-H19 transfection or knockdown under normoxia and H/SD conditions. [score:2]
Alterations of VEGFA in different experimental groups were detected after miR-199a-5p transfection or knockdown under normoxia and H/SD condtions. [score:2]
a Potential binding sites of lncRNA-H19 and miR-199a-5p. [score:1]
Further exploration of the pleiotropic effects of lncRNA-H19 and the crosstalk between lncRNA-H19 and miR-199a-5p will provide new insights for developing new strategies to improve the therapeutic efficacy based on MSCs transplantation. [score:1]
Blank control, MSCs transfected with luciferase reporter containing H19-WT or H19-MU; miR-199a-5p, MSCs cotransfected with miR-199a-5p mimics and luciferase reporter containing H19-WT or H19-MU; miR-199a-5p NC, MSCs cotransfected with miR-199a-5p mimics negative control and luciferase reporter containing H19-WT or H19-MU. [score:1]
It was indicated that cotransfection of psiCHECK2-H19-WT and miR-199a-5p mimics distinctly diminished the relative luciferase activity in contrast with that in the blank control and miR-199a-5p NC groups (P < 0.01, Fig.   7b), while no difference was revealed in the relative luciferase activity between the psiCHECK2-H19-MU+miR-199a-5p and psiCHECK2-H19-MU+miR-199a-5p NC groups (P < 0.01, Fig.   7b). [score:1]
Bioinformatic prediction combined with functional experiments proffered a further sustained interaction between lncRNA-H19 and miR-199a-5p. [score:1]
Nevertheless, the MSCs+si-H19 group showed a significantly elevated level of miR-199-5p in contrast to the MSCs+si-H19 NC and MSCs groups (P < 0.01, Fig.   7c). [score:1]
Cells were seeded in 24-well plates and cotransfected with wild-type or mutated luciferase construct along with miR-199a-5p mimics or miR-199a-5p mimics negative control. [score:1]
Two luciferase reporters containing wild-type H19 (psiCHECK2-H19-WT, which encompassed the binding sites for miR-199a-5p) or mutant H19 (psiCHECK2-H19-MU, which encompassed the mutant sequence of the binding sites for miR-199a-5p) were constructed to validate the interaction between H19 and miR-199a-5p. [score:1]
MiR-199a-5p plays a negative regulatory role in cell survival and angiogenesis. [score:1]
MiR-199a-5p negatively regulated VEGFA. [score:1]
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[+] score: 159
miR-199a-3p inhibits angiogenesis by directly reducing VEGF secretion from CACs and suppressing expression of its receptors VEGFR1 and VEGFR2 on ECs. [score:8]
[32] The tumor suppressor miRNA, miR-199a-3p is downregulated in HCC liver tissues as observed in several studies and its decrement significantly correlates with poor prognosis and survival of the patients, whereas restoration of miR-199a-3p in HCC cell line leads to reduced invasiveness and enhanced doxorubicin sensitivity by abrogating mTOR, CD44, and cMET proto-oncogene. [score:6]
In addition to the elucidation of the mechanism of HCC development, a single small noncoding multipotent tumor suppressor miRNA, miR-199a-3p, has been identified in this study using both in vitro and in vivo assays, which has robust potential to regulate multiple proteins involved in the intercellular signaling network executed during progression of liver diseases to HCC. [score:6]
Thus these findings suggest that miR-199a-3p may repress migration and angiogenesis of ECs by inhibiting the secretion of VEGFA from HCC cells and subsequently abrogating the pro-angiogenic signaling pathway by targeting VEGFR2 on ECs. [score:5]
In 2010, Fornari et al. [28] had shown that miR-199a-3p inhibits HGF/cMET signaling by targeting MET in HCC. [score:5]
Collectively, our data signify that miR-199a-3p is a potent multifaceted tumor suppressor miRNA, which attenuates intercellular communications between HSCs–CACs and CACs–ECs, thereby inhibiting signal initiation, migration, metastasis and angiogenesis in HCC. [score:5]
Furthermore, a dramatically reduced level of angiogenesis was noted either on overexpression of miR-199a-3p in ECs in the presence of recombinant VEGFA or addition of anti-VEGFA antibody -treated CM of SNU449 on HUVEC (Figure 3f) implicating that miR-199a-3p attenuated expression of VEGFR2 on ECs. [score:5]
Immunoblotting and confocal microscopy analysis in the presence or absence of miR-199a-3p consistently depicted that this miRNA directly regulated the expression of HGF (Figures 5a, b and Supplementary Figure S4). [score:5]
To understand the molecular mechanism of inhibition of angiogenesis, we used a systemic approach to identify molecular targets of miR-199a-3p. [score:5]
Thus restoration of miR-199a-3p in ECs might suppress neoangiogenesis by inhibiting downstream angiogenic signaling pathways. [score:5]
To understand the molecular mechanism by which miR-199a-3p inhibited tumor metastasis, one of the most attractive metastatic genes, hepatocyte growth factor (HGF) identified in bioinformatics analysis as its' target (Supplementary Table 1) was validated in vitro. [score:5]
Inhibition of the intracellular protein expression of HGF by miR-199a-3p also reduced the secretion of HGF in the culture media of LX2 cells as confirmed by ELISA (Figure 5c). [score:5]
Thus miR-199a-3p targets VEGFR1, VEGFR2 and probably PDGFR α on ECs and soluble factor VEGFA and ANGPT1 secreted from CACs to inhibit angiogenesis. [score:5]
Co-culturing of ECs with HCC cells pre -transfected with premiR-199a-3p significantly reduced the migration ability of ECs in transwell assay, whereas restoration of miR-199a-3p expression in ECs suppressed the tube-formation ability in the presence of VEGF growth factor. [score:4]
miR-199a-3p is downregulated in HCC tissues. [score:4]
Moreover, blockage of HGF expression by shRNA mimics the effect of miR-199a-3p whereas overexpression of HGF was considered as positive control in western blot, confocal analysis and ELISA (Figures 5a, b and Supplementary Figure S4). [score:4]
Among these genes, miR-199a-3p downregulated mostly the pro-angiogenic factor VEGFA, which is secreted from CACs and its receptors VEGFR1, VEGFR2 located on ECs. [score:4]
Hepatocyte-stellate cells cross talk inhibited by miR-199a-3p attenuating invasion and metastasis of CACs. [score:3]
miR-199a-3p or vector overexpressed SNU449 (1 × 10 [6]) cells with matrigel (BD Biosciences, San Jose, CA, USA) at 1:1 dilution were injected subcutaneously into the right flank region of NOD/SCID mice (four mice in each group). [score:3]
Ectopic expression of mature miR-199a-3p at different time points was confirmed after transfecting the premiR-199a-3p plasmid in HepG2 cells by qRT-PCR (Figure 3b). [score:3]
These findings suggested that miR-199a-3p inhibited VEGF secretion from CACs and also attenuated its receptor -mediated downstream signaling via VEGFR2. [score:3]
Thus miR-199a-3p inhibits invasion, migration and angiogenesis by reducing genes on both CACs and ECs and hence disrupts communication between them. [score:3]
miR-199a-3p inhibits tumor angiogenesis and migration by attenuating cross talk between CACs and ECs. [score:3]
Moreover, the introduction of miR-199a-3p diminished the expression of MMP2 as observed in western blot analysis and its activity in gelatin zymography suggesting that miR-199a-3p exerts its effect by binding to the 3′-UTR of MMP2 gene (Figures 6c and d). [score:3]
Phospho-thiorate modified perfect complementary oligo [5′-TAACCAATGTGCAGACTACTGT-3′ modified in first two bases] was used to inhibit miR-199a-3p. [score:3]
To examine the anti-tumorigenic role of miR-199a-3p, premiR-199a-3p overexpressing SNU449 cells (Supplementary Figure S1) were injected subcutaneously (s. c. ) into the right flank of NOD/SCID mice. [score:3]
miR-199a-3p and backbone vector overexpressing SNU449 cell lines were generated by G418 selection after transfecting SNU449 cells with premiR-199a-3p and vector plasmid, respectively. [score:3]
These evidences strongly suggested that miR-199a-3p represses the cross talk between HSCs and CACs and thus inhibits invasion and migration of CACs. [score:3]
Similarly, lower number of ECs was migrated on restoration of miR-199a-3p expression within HCC cells in co-culture condition (Figures 2a and b). [score:3]
For metastatic mo del, miR-199a-3p or vector overexpressed SNU449 (1 × 10 [6]) cells were injected through lateral tail vein of NOD/SCID mice (four mice in each group), and after 4 weeks, the mice were killed and the number of nodules in the lung was counted and the lung tissue was stained with hematoxylin and eosin. [score:3]
[28] In our study, HGF was identified as a potent target of miR-199a-3p and co-culturing of HCC cells with premiR-199a-3p -transfected HSCs dramatically attenuated migration and invasion of HCC cells through matrigel by abrogating the cross talk between HSCs and CACs. [score:3]
The gain of function analysis of mir-199a-3p in respective cell lines had further confirmed that this miRNA diminished the expression of intracellular VEGFA in SNU449 and VEGFR2 in HUVEC as observed by western blot analysis (Figure 3c). [score:3]
In vivo restoration of miR-199a-3p shows anti-tumorigenic activity associated with reduced angiogenesisTo examine the anti-tumorigenic role of miR-199a-3p, premiR-199a-3p overexpressing SNU449 cells (Supplementary Figure S1) were injected subcutaneously (s. c. ) into the right flank of NOD/SCID mice. [score:3]
Tumor growth was suppressed in the presence of miR-199a-3p as compared with vector control (Figures 1d and e). [score:2]
In this study, ANGPT1 has been identified as a target of miR-199a-3p in bioinformatics analysis and 3′-UTR luciferase-reporter assay. [score:2]
miR-199a-3p exerts its effect by negatively regulating HGF signaling. [score:2]
[29] Dual reporter luciferase assay with 3′-UTR construct showed that overexpression of miR-199a-3p significantly reduced the luciferase activity (Figure 6a). [score:2]
Binding aptitude of miR-199a-3p to the target gene was determined by luciferase-reporter assay using 3′-UTR of VEGFA, HGF, VEGFR1, VEGFR2, ANGPT1 and MMP2 cloned in psiCHECK2 vector (Promega, Madison, WI, USA) and transfected in HepG2 cells. [score:2]
Bioinformatics analysis and luciferase-reporter assay indicated that the receptor of PDGF, PDGFR α, located on HSCs and ECs was another important target of miR-199a-3p (data not shown). [score:2]
Thus, by favorably modulating specific regulators of intercellular signaling pathways and components of TME, miR-199a-3p offers fascinating therapeutic potential for HCC. [score:2]
org), we found the complementary sequences of miR-199a-3p in the 3′-UTR of four important genes of angiogenesis VEGFA, VEGFR1, VEGFR2 and ANGPT1 (Supplementary Table 1). [score:1]
We further explored the molecular mechanisms of multifaceted role exerted by miR-199a-3p. [score:1]
In vivo restoration of miR-199a-3p shows anti-tumorigenic activity associated with reduced angiogenesis. [score:1]
VEGF–VEGFR signaling genes were further validated with miRNA binding-site mutants of miR-199a-3p (Figure 3a). [score:1]
Thus, reduced migration and invasion ability of HCC cells (SNU449) when co-cultured with premiR-199a-3p -transfected HSCs (LX2) revealed that miR-199a-3p could diminish both HGF and cMET and thus abrogate the generation of signal in HSCs and its transmission into CACs. [score:1]
Thus interfering appropriately with various routes of cross talk of CACs and stromal components of TME, mir-199a-3p has opened a new therapeutic avenue for the effective treatment of HCC patients. [score:1]
MMP2 signaling is also abrogated by miR-199a-3p. [score:1]
Extracellular VEGFA level in the media of HCC cell line was also reduced in the presence of miR-199a-3p and it was reverted back to the normal level when treated with anti-miR-199a-3p as observed by ELISA (Figure 3d). [score:1]
miR-199a-3p also blocks downstream signaling pathways in ECs. [score:1]
with full-length 3′-UTR construct consisting of three miR-199a-3p binding sites revealed that the co-transfection of miR-199a-3p significantly reduced the luciferase activity to 45%, which was recovered to 83% with single miRNA binding-site mutant construct where two other binding sites were unaltered. [score:1]
39, 40 An inverse relation in cMET and miR-199a-3p has been reported in HCC recently. [score:1]
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[+] score: 157
Since virtually 100% HCCs exhibit a strong down-regulation of miR-199 which is instead expressed at substantial level in normal hepatocytes, where it represents the third most highly expressed miRNA [48], this virus was designed to prevent cytolytic activity in healthy cells, thereby addressing the important issue of a novel therapeutic approach with improved efficacy and safety in HCC. [score:8]
This evidence confirms that miR-199, constitutively expressed in HepG2/199 cells, can control E1A expression by targeting the homologous sequences places downstream of the adenoviral E1A gene. [score:7]
TaqMan, Real Time PCR analysis showed that miR-199 expression was significantly increased in the HepG2/199 cell line in comparison with the basal expression level in the HepG2 cells (p-value = 0.0005) and not significantly different from human normal liver (NL) expression levels (p-value = 0.06). [score:7]
The inhibition of E1A mRNA and protein was demonstrated in miR-199 expressing HepG2 cells, while E1A normal expression could be detected in HepG2 wild type cells (Figure 2A-B ). [score:7]
The rationale of the work was based on the differential expression of miR-199 between normal versus cancer liver cells and in particular on the basis that miR-199 is down-regulated in human hepatocellular carcinoma [12]. [score:6]
To verify if miR-199 could regulate viral replication in vitro, Ad-199T and Ad-Control were used to infect two different cell lines: (1) HepG2, derived from human liver carcinoma and not expressing miR-199; (2) HepG2/199, which derives from HepG2 cells engineered to constitutively express miR-199a (Figure S3 ). [score:6]
To produce stable cell clones expressing miR199, HepG2 cells were transfected with 2 µg of a miR-199 expressing plasmid, pIRES-miR199, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). [score:5]
This objective was achieved by introducing into the viral genome multiple miR-199 target sites able to modulate the expression of the E1A gene. [score:5]
Quantitative PCR revealed that miR-199 was down-regulated in tumors in comparison with normal liver (p value = 0.0146) (Figure 8E ). [score:4]
In particular, miR-199 was reported to be consistently down-regulated in HCC [12]. [score:4]
Useful for the objective of this study, we previously showed that miR-199 is down-regulated in liver tumors arising in this mo del. [score:4]
The involvement of miR-199 in the pathogenesis of HCC was linked to the abnormal regulation of multiple target genes, such as mTOR, c-Met, HIF-1α and CD44 [13, 14, 15, 16]. [score:4]
This mouse strain has an increased susceptibility to the carcinogen diethylnitrosammine (DEN) and tumors exhibit a miRNA profile similar to human HCC, including the down-regulation of miR-199. [score:4]
This latter vector was used as recipient of the miR-199 targeting site (199T) into the MluI restriction site, to generate the pENTR_E1A/199T/E1B vector. [score:3]
As expected, the luciferase activity was significantly decreased only in samples co -transfected with miR-199 (p value = 0.007), thus proving the functional interaction between miR-199 and the artificial target sequences (Figure S1 ). [score:3]
By investigating in vitro and in vivo properties of Ad-199T, we demonstrated that this CRAd could replicate very poorly in cells expressing miR-199, while its replication could proceed regularly in cells lacking the expression of this miRNA. [score:3]
HepG2/199 cell line stably express miR-199. [score:3]
For the construction of the adenoviral miR-199 dependent plasmid, an MluI fragment containing the microRNA-199 target sequence was amplified by PCR from the plasmid pGL3-199T and inserted into a MluI site located in the 3’ UTR of E1A sequence in the vector pENTR_E1A/E1B, to generate pENTR_E1A/199T/E1B. [score:3]
E1A expression is miR-199 dependent. [score:3]
0073964.g002 Figure 2To assess the expression of E1A in presence or absence of miR-199, 7×10 [4] cells of HepG2 and HepG2/199 cell lines were seeded and infected with 1x10 [6] I. U. of Ad-199T or with 1x10 [6] I. U. of Ad-Control and harvested after 48 hours. [score:3]
A replicative control adenovirus, Ad-Control, without miR-199 target sequences, was also developed (Figure 1 ). [score:3]
This work establishes that the presence of miR-199 target sites within the 3’ UTR of E1A gene represents a strategy to generate recombinant adenoviruses with significant oncolytic activity against liver cancer, which exhibits low level of miR-199, coupled with reduced hepatoxicity. [score:3]
This foreseeable finding was a consequence of the inhibitory effect imposed by miR-199 on Ad-199T replication, thereby preventing its lytic activity in healthy cells. [score:3]
To assess if these findings were related to miR-199 expression, we analyzed the level of the miRNA in normal liver and tumor tissues. [score:3]
First, in 3 days old mice, which have a limited or absent immune response [49], Ad-199T was not able to replicate in the liver while an identical control virus, lacking the miR-199 target sites, could efficiently undergo several rounds of replication. [score:3]
A progressive accumulation of viral DNA in the HepG2 cells infected with Ad-199T indicates that active viral replication is efficiently occurring in this cell line; on the contrary, in HepG2/199 cells infected with Ad-199T the viral DNA did not increase over time, confirming that the virus replication was inhibited by the presence of miR-199. [score:3]
Figure S9(A) To asses miR-199 expression levels in Hep3B cell line, a TaqMan, Real Time PCR was performed. [score:3]
The present work is the first that make use of miR-199 to target an oncolytic virus to cancer cells in vivo. [score:3]
The results showed that Hep3B displayed a very low basal miR-199 expression level, even lower than HepG2 cells. [score:3]
To confirm its functional activity, we first cloned the miR-199 artificial target sequence downstream of the firefly luciferase reporter gene at the XbaI restriction site within the pGL3 vector (Promega, Madison, WI, USA), to generate pGL3/199T vector. [score:3]
Figure S3 The pIRES-miR199 vector, expressing miR-199, was stably transfected in the hepatocellular carcinoma derived cell line HepG2, generating the HepG2/199 cell line. [score:3]
Since miR-199 is instead expressed at variable but significant level in any normal tissues, Ad-199T could lack toxicity in tissues other than liver as well. [score:3]
Since miR-199 is highly expressed in normal liver, but not in HCC, this virus seems to be well suited for the treatment of liver cancer. [score:3]
To assess replication properties of Ad-199T in vivo, we tested its ability to replicate in the liver of B6D2 wild type mice, where miR-199 is constitutively expressed. [score:3]
The MluI site was used for introducing the miR-199 target segment, originally cloned into the pGL3/199T (as described above). [score:3]
To assess the expression of E1A in presence or absence of miR-199, 7×10 [4] cells of HepG2 and HepG2/199 cell lines were seeded and infected with 1x10 [6] I. U. of Ad-199T or with 1x10 [6] I. U. of Ad-Control and harvested after 48 hours. [score:3]
miR-199 directly interacts with its target sequence cloned in the pGL3/199T vector, as evaluated by luciferase activity in Hep3B cells. [score:2]
These results established that viral replication of Ad-199T was indeed miR-199 -dependent in vitro. [score:1]
Four copies of a 22 bp DNA segment complementary to miR199 were inserted within the 3’-untraslated region (3’-UTR) of the E1A gene, which is essential for adenoviral replication. [score:1]
To develop a conditionally replication-competent oncolytic adenovirus (CRAd) under miR-199 control, named Ad-199T, four copies of a 22 bp DNA segment complementary to human and mouse miR-199, were inserted within the 3’-untraslated region (3’-UTR) of the E1A gene, essential for adenoviral replication. [score:1]
Here, to improve tumor specificity and reduce toxicity for normal cells, we developed a novel CRAd whose replication was controlled by miR-199. [score:1]
This vector was transfected into the hepatocarcinoma Hep3B cells together with either miR-199 or scrambled oligos. [score:1]
The pre-miR-199a-3p miRNA precursor (Ambion Applied Biosystems, Grand Island, NY, USA) or the control oligonucleotide (AM17111, Ambion Applied Biosystems, Grand Island, NY, USA) were co -transfected at a concentration of 100 nM. [score:1]
These results demonstrated that Ad-Control replicates efficiently in normal liver cells, inducing hepatotoxicity, while miR-199 could control Ad-199T lytic cycle in normal hepatocytes in vivo. [score:1]
This evidence confirms that miR-199 could control Ad-199T replication in normal liver cells in vivo. [score:1]
Taken together, these findings can be interpreted as evidence that Ad-199T could replicate more efficiently in tumor tissues, where it can produce cytolysis, and this property was most likely miR-199 -dependent. [score:1]
Here, we took advantage of this information to produce a new type of CRAd able to replicate only in cells lacking miR-199, with the aim of making viral replication and cytolytic effect specifically selective for HCC cells. [score:1]
The Firefly Luciferase reporter activity was significantly decreased when pGL3/199T vector was co -transfected with the pre-miR-199a-3p miRNA precursor (p value = 0.007). [score:1]
Figure S1The Firefly Luciferase reporter activity was significantly decreased when pGL3/199T vector was co -transfected with the pre-miR-199a-3p miRNA precursor (p value = 0.007). [score:1]
The Ad-199T virus was designed to replicate in miR-199 -negative cells. [score:1]
miR-199 controls Ad-199T replication in vivo. [score:1]
miR-199 controls Ad-199T replication in vitro. [score:1]
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[+] score: 156
In summary, our findings revealed that ER stress suppresses the expression of the miR-199a/214 cluster by activating NFκB to upregulate pro-survival XBP-1 expression, which suggested a novel UPR/NFκB/miR-214/XBP-1 regulatory circuitry whose dysfunction may contribute to tumor survival and progression of HCC. [score:11]
To start unraveling the regulatory mechanisms of miR-199a2/214 expression under UPR conditions in greater detail, we further found that UPR activated NFκB with concomitant suppression of miR-199a2/214 transcription, and this suppression was reversed by NFκB inhibitor PDTC in HepG2 cells, which suggested that NFκB is a potential negative regulator of the miR-199a-2/miR-214 cluster. [score:11]
We further identified that NFκB activated by unfolded protein response (UPR) suppresses miR-199a2/214 transcription, and demonstrated that activation of UPR and endoplasmic reticulum (ER) stress represents an important mechanism responsible for miR-214 and miR-199a-3p/5p down-regulation in HCC development. [score:7]
To further understand the mechanism of the miR-199a/214 cluster down -expression in HCC, we found that thapsigargin (TG) and tunicamycin (TM) or hypoxia -induced unfolded protein response (UPR) suppresses the expression of the miR-199a/214 cluster in HCC cells. [score:7]
Decreased miR-214 expression was observed in 65% of HCC (15 of 23 cases), and consistent down-regulation of both miR-199a-3p and miR-199a-5p also were detected in as much as 73% of HCC (17 of 23 cases) (Figure 1A and B). [score:6]
In our study, we showed that NFκB and XBP-1 were predominantly expressed but miR-214 was significantly reduced in human HCC tissues, miR-214 directly targets XBP-1, and UPR or hypoxia induced-NFκB activation negatively controls the miR-199a/214 cluster transcription in HCC cells. [score:6]
Result show that miR-214 and miR-199a-3p/5p was significantly down-regulated in HepG2 cells after TG and TM treatments or anoxia, further suggesting that UPR activated XBP-1 or mTOR and ERK pathway to protect tumor cell survival though suppression of the miR-199a2/214 cluster in HCC. [score:6]
Unfolded protein response downregulates miR-199a/214 expression in HCC cells. [score:6]
Thus, miR-199a and miR-214 expression levels are down-regulated by UPR under various physiological and pathological conditions. [score:6]
In the other hand, our study showed that a significant down-regulation of the miR-199a/214 cluster was observed in human HCC tissues and HCC cell lines when compared with normal liver, consistent with previous observations from profiling of miRNAs expression in HCC [9], [39], [40], [41], [42]. [score:5]
0031518.g005 Figure 5(A) HepG2 cells treated with Thapsigargin(TG, 5 µmol/L) and tunicamycin (TM, 5 µg/ml) for 24 h were analyzed by western blotting for GRP94 and XBP1 expression levels and analyzed by real-time RT-PCR for miR-199a-3p/-5p and miR-214 expression. [score:5]
0031518.g006 Figure 6(A) LPS (10 µg/ml) treatment induced NFkB p65 expression, attenuates the miR-199a/214 expression in SMMC-7721 cells. [score:5]
As the UPR transcription factor XBP-1 was identified as a target of miR-214 and recent studies have revealed the important functions of miR-199a/b-3p in HCC carcinogenesis and progression by targeting mTOR and c-Met or PAK4/Raf/MEK/ERK Pathway in HCC cells [5], [21], we decided to further investigate the correlation between UPR activation and miR-199a/214 down -expression. [score:5]
Our study suggest that modulation of miR-214 levels may provide a new therapeutic approach for cancer treatment and revealed that UPR may offer a new explanation for why the miR-199a/214 cluster were down-regulated in the progression in HCC. [score:4]
Further, we also explored whether NFκB is able to regulate miR-199a/214 expression upon anoxia. [score:4]
Therefore, a new UPR/NFκB/miR-214/XBP-1 regulatory circuitry was suggested in HCC progression, in which NFκB was activated by UPR and participated in the negative regulation of miR-199a/214 to regulate HCC progression (Figure 7). [score:4]
miR-199a/214 is downregulated in HCC cell lines and tissues. [score:4]
The regulatory role of NFκB in miR-199a/214 Expression. [score:4]
ER stress induces miR-199a/214 downregulation in HCC cells. [score:4]
showed that these miRNAs were all significantly down-regulated and miR-199a-3p>miR-199a-5p>miR-214 compared with adjacent nontumorous liver tissues. [score:3]
In the present study, we showed that miR-199a-3p, miR-199a-5p and miR-214 expression was significantly reduced in HCC tissues. [score:3]
The expression levels of miR-199a and miR-214 were also lower in HCC cells exposed to anoxia induced by CoCl [2] (100 µmol/L) (Figure 5B). [score:3]
miR-199a/214 expression in HCC samples and cell lines. [score:3]
But what is the mechanism of the miR-199a/214 cluster down -expression in HCC? [score:3]
0031518.g001 Figure 1(A) Box plot graphic displays of 23 HCC and matched adjacent benign tissues grouped according to miR-199a-3p and miR-199a-5p expression. [score:3]
Our study offer new insight into the tumor suppressor activity conferred by miR-199a/214 and the potential mechanisms of hepatocarcinogenesis. [score:3]
In addition, we further tested the effect of hypoxia -induced UPR on the level of miR-199a/214 expression. [score:3]
Some particular miRNAs were reported to be differentially expressed in different cancer, such as the miR-199a/214 cluster. [score:3]
In parallel, in HCC cell lines HepG2 and SMMC-7721, miR-199a-3p/5p and miR-214 expression was markedly decreased compared with that in human normal liver (Figure 1C). [score:2]
NFκB is a potential negative regulator of the miR-199a-2/miR-214 gene. [score:2]
Summary diagram describes the ER stress/NFκB/miR-199a/214 network that regulates HCC tumorigenicity. [score:2]
Certainly, more evidences of NFκB bind site in the promoter of the miR-199a/214 cluster are needed in future to support this hypothesis and more investigations are needed to elucidate whether ER stress also activate other factors (e. g. Sp1) together involved in the downregulation of miR-199a/214 in HCC. [score:2]
These data indicated that NFκB is a potential negative regulator of the miR-199a-2/miR-214 cluster. [score:2]
Furthermore, the miPPR-199a2 region is shown here to be the authentic miR-199a2 promoter that produces the primary transcript harboring the miR-199a-3p, miR-199a-5p and miR-214 sequences as a cluster [25]. [score:1]
The miR-214, miR-199a-3p and miR-199a-5p level was quantified by real-time quantitative-PCR using TransStartTM SYBR Green qPCR Supermix (TransGen Biotech, Beijing, China), and with U6 small nuclear RNA as an internal normalized reference. [score:1]
The miR-214/XBP-1 pathway was shown in this work, while miR-199a-3p/mTOR and miR-199a-5p/DDR1 pathways were reported by other studies. [score:1]
Although miR-199a-3p and miR-199a-5p have been reported to contribute to liver carcinogenesis [5], [21], [22], the role of miR-214 in HCC tumorigenesis has not been elucidated. [score:1]
0031518.g007 Figure 7 The miR-214/XBP-1 pathway was shown in this work, while miR-199a-3p/mTOR and miR-199a-5p/DDR1 pathways were reported by other studies. [score:1]
Recently, miR-199a-3p/5p was verified to be decreased in HCC tissues, and its decrement significantly correlates with the survival of HCC patients, outlining a potential marker for predicting the prognosis of HCC patients [5], [21], [22]. [score:1]
Nevertheless, further understanding of the molecular mechanism and network by which the miR-199a/214 cluster functions may provide new avenues of research that could aid early diagnosis and treatment of this highly malignant tumor. [score:1]
It is well known that there are two genes that potentially encode pri-miR-199a, the primary precursor of hsa-mir-199a. [score:1]
To study the role of the miR-199a/214 cluster in the HCC, levels of miR-214 and miR-199a-3p/5p were determined in 23 pairs of HCC and adjacent benign tissues using real-time PCR. [score:1]
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[+] score: 140
TGF-β1 induced expression of p53, latter increased expression of miR-199a-3p to suppress SOCS7 expression and subsequently promote activation of STAT3 and ECM accumulation. [score:9]
Furthermore, miR-199a-3p directly suppressed SOCS7 expression, which led to activation of STAT3 and upregulation of the production of probrotic proteins. [score:9]
Collectively, these data suggest a novel regulatory mechanism by which p53 upregulates STAT3 activation by direct inducing miR199a-3p to suppress SOCS7 in HK-2 cells (Fig. 11). [score:8]
In addition, we have revealed that p53 induces STAT3 activation to promote renal fibrosis via direct upregulation of miR199a-3p to suppress SOCS7 in HK-2 cells. [score:7]
To provide more direct evidence of miR-199a-3p targeting SOCS7, we co -transfected HK2 cells with a plasmid containing a luciferase report gene under the control of SOCS7 3′ untranslated region (UTR) and either a miR-199a-3p analog or a miRNA analog negative control. [score:6]
Previous results demonstrated that p53 directly induced miR-215 expression 33, which is known to be involved in increased collagen production and the progression of diabetic nephropathy by regulating the CTNNBIP1/β-catenin pathway 20. miR-199a-5p (previously called miR-199a) and miR-199a-3p (previously called miR-199a*) are two mature forms derived from the same precursor in the human genome 34 35. [score:5]
The expression of miR-215-5p, miR-199a-5p & 3p was analyzed by real-time PCR and Northern blot (Fig. 7C–F), demonstrating that the induction of miR-215, miR-199a-5p&3p after UUO was inhibited in PT-p53- KO mice. [score:5]
Pifithrin-α suppressed TGF-β1 -induced STAT3 activation and miR-199a-3p expression. [score:5]
We identified SOCS7 as the target of miR-199a-3p but not 5p predicted by the TargetScan database (http://www. [score:5]
In HK-2 cells and mouse mo dels, inhibition of p53 suppressed STAT3 activation, a key signaling of renal fibrosis, by inducing miR199a-3p that represses SOCS7. [score:5]
Furthermore, our luciferase reporter assay identified SOCS7, one member of the family of suppressors of cytokine signaling (SOCS), as a target gene of miR-199a-3p in HK-2 cells. [score:4]
The expressions of three miRNAs, miR-215-5p and miR-199a-5p&3p, were found to be consistently high in wide type mice with UUO, and they were decreased in renal cortex of global p53 knockout mice with UUO. [score:4]
However, the two down-regulated miRNAs, miR-215-5p and miR-199a-5p were correlated with the amelioration of renal fibrosis 20 21. [score:4]
A recent study reported that miR-199a-5p suppressed caveolin1 for CCl4 -induced liver fibrosis and UUO -induced renal fibrosis 21. [score:3]
We proposed that miR-199a-3p could promote STAT3 activation by inhibiting SOCS7. [score:3]
We found that the expression of fibronectin, collagen I, α-SMA, STAT3 tyrosine phosphorylation (Tyr705), and miR199a-3p during UUO injury was markedly reduced in PT-p53- KO kidney tissues than in the wild-type tissues (Fig. 10G–I). [score:3]
The induction of miR-215, miR-199a-5p, and miR-199a-3p after UUO was suppressed in PT-p53- KO mice. [score:3]
The induction of miR-215, miR-199a-5p&3p after UUO was suppressed in PT-p53- KO mice. [score:3]
The results indicated that TGF-β1 treatment markedly increased expression of vimentin and collagen I, which was significantly enhanced by transfection with miR-199a-3p analog (Fig. 8D,E). [score:3]
Furthermore, we detected that miR199a-3p expression in IgAN and DN was higher than in kidneys with MCD (Fig. 11H). [score:3]
How to cite this article: Yang, R. et al. p53 induces miR199a-3p to suppress SOCS7 for STAT3 activation and renal fibrosis in UUO. [score:3]
Data were expressed as means ± sd (n = 6); [#] p <  0.05: miR-199a-3p vs miR-ANC, or SiRNA SOCS7 vs control group. [score:3]
We found that TGF-β1 treatment markedly increased activation of STAT3 and the expression of collagen I, vimentin, and miR-199a-3p, and reduced SOCS7 protein, which was significantly reversed by pifithrin-α treatment (Fig. 8F–H). [score:3]
TGF-β1 increased miR-199a-3p expression by induction of p53. [score:3]
The results revealed that p53 induced miR-199a-3p to suppress SOCS7 for STAT3 activation. [score:3]
As shown in Fig. 9F, the antibody directed against p53 immunoprecipitated the DNA fragments from HK-2 cells containing the potential binding sites of pBS1 and pBS2, supporting the hypothesis that p53 can physically interact with the miR-199a-3p promoter region. [score:2]
MiR-199a-3p&5p were mature forms of mmu-miR-199a, hence, we further presumed that miR-199a-3p could be involved in the regulation of renal fibrosis like miR-199a-5p. [score:2]
MiR-199a-3p suppressed SOCS7 to increase STAT3 activation. [score:2]
MiR-199a-3p suppressed SOCS7 for STAT3 activation and renal fibrosis. [score:2]
However, the regulation mechanism of miR-199a-3p for fibrosis remains unclear. [score:2]
A recent study reported that miR-199a-5p was regulated by TGF-β primarily through a Smad -dependent post-transcriptional mechanism promoting miRNA maturation by Drosha 42. [score:2]
For Pifithrin-a treatment, HK-2 cells were treated with or without Pifithrin-a (10 μM) or TGF-β1 (10 ng/ml) for 24 h. For transfection experiment, after 24 h transfection of miR-199a-3p analog (100 nM) or negative control (miR-neg, Sigma) or SOCS7 siRNA (sc-41004, Santa Cruz, CA, USA). [score:1]
In current study, we found that miR-199a-3p was also a key effector of TGFβ signaling in HK-2 cells. [score:1]
How about the role of miR199a-3p in the progression of fibrosis? [score:1]
Previous studies have demonstrated that both miR-215-5p and miR-199a-5p were related to the renal fibrosis 20 21, hence, we focused on the miR-199a-3p. [score:1]
The mechanisms involved in the TGFβ -dependent modulation of miR-199a-3p are also of particular interest. [score:1]
Immunoblot (A) analysis of p-p53 Ser 15 and p53, densitometry (B) of proteins signals on immunoblots, and real time PCR analysis (C) of miR-199a-3p after indicated time point of treatment with TGF-β1. [score:1]
Total RNA (10 μg per lane) was analyzed by Northern blotting as described in the concise Methods section using a [32]p-labeled probe of miR-215, miR-199a-5p, and miR-199a-3p. [score:1]
Relative protein levels (D) of vimentin, COL1, and β-actin 24 h after transfection of miR-199a-3p analog (100 nM) or miR analog negative control (miR-ANC) with or without TGF-β1 treatment, densitometry (E) of proteins signals on immunoblots. [score:1]
First, the real time PCR results demonstrated that miR-199a-3p was significantly induced by TGF-β1 at 2 h and 24 h (Fig. 8C). [score:1]
The lysate of the kidney cortex was collected for immunoblot analysis of p53, p-STAT3 (Tyr705), fibronectin, collagen I, α-SMA, and β-actin by using specific antibodies (E, G), densitometry (F, H) of proteins signals on immunoblots, and real time PCR analysis of miR-199a-3p (I). [score:1]
Immunoblot analysis (F) of vimentin, COL1, SOCS7, p-STAT3, STAT3 and β-actin, densitometry (G) of proteins signals on immunoblots, and real time PCR analysis (H) of miR-199a-3p 24 h after TGF-β1 alone or TGF-β1 plus pifithrin-a treatment. [score:1]
In the pathogenesis of tissue fibrosis, both miR-199a-5p and miR-199a-3p were associated with the progression of liver fibrosis in humans and mice 39 40. [score:1]
Real time PCR analysis of miR-199a-3p (H). [score:1]
Cultured HK-2 cells were treated with transfection of miR-199a-3p or negative control analog or SOCS7 siRNA, followed by immunoblot for p-STAT3, STAT3, and ECM genes, and immunoprecipitation with antibodies to p53. [score:1]
Cultured HK-2 cells were treated with 10 ng/ml TGF-β1 or 10 μM pifithrin-a for 0 to 24 h or transfection of miR-199a-3p or negative control analog or SOCS7 siRNA, followed by immunoblot for p-STAT3, STAT3, and ECM genes, and real time PCR for miR-199a-3p. [score:1]
As shown in Fig. 9A,B, transfection of HK2 cells with miR-199a-3p also led to significant decrease in SOCS7 protein. [score:1]
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[+] score: 109
Because miR-199a-3p regulates translation of both versican and fibronectin, transcription of versican will share the load of suppression and allow increased translation of both versican and fibronectin [24]. [score:8]
The miR-199a-3p target site was highly conserved in human and mouse genome, suggesting its importance in regulating Rb1 translation. [score:6]
Two miRNAs (miR-199a-3p and miR-144) that have been shown to interact with versican 3′UTR [24] were hypothesized to inhibit Rb1 translation (SI Figure S4a). [score:5]
The presence of a mutated binding site alleviated luciferase activity from translational inhibition by miR-199a-3p and restored luciferase activity by 20%. [score:5]
Based on the results of Rb1 targeted by miR-144 and miR-199a-3p, it is anticipated that other miRNAs targeting Rb1 could function in the similar fashion. [score:5]
Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3′UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. [score:5]
In mice, miR-199a-3p has another target site located nearby the first confirmed target site (SI Figure S5a). [score:5]
Rb1 expression is regulated by miR-199a-3p and miR-144. [score:4]
Currently, miR-199a-3p is the only miRNA that has been shown to regulate versican expression by binding to its 3′UTR. [score:4]
Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. [score:4]
In our previous study, we have shown that versican 3′UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. [score:4]
The target sequences of miR-199a-3p and miR-144 in the Rb1 3′UTR are conserved in human and mice. [score:3]
Computational analysis showed that miR-199a-3p, miR-144, and miR-136 potentially targeted versican 3′UTR. [score:3]
We found that expression of versican 3′UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. [score:3]
0013599.g006 Figure 6 Computational analysis showed that miR-199a-3p, miR-144, and miR-136 potentially targeted versican 3′UTR. [score:3]
Since miR-199a-3p has been shown to target versican and fibronectin [24], tumor sections were stained with anti-versican and anti-fibronectin antibodies. [score:3]
We reasoned that different tissues might exhibit different levels of miR-199a-3p expression, but more importantly, different tissues might respond differently to the presence of 3′UTR. [score:3]
Overexpression of versican 3′UTR would attract endogenous miR-199a-3p, miR-144, and miR-136. [score:3]
The expression of miR-199a-3p and miR-136 were detected in both cells and several primary tissues but miR-144 was not found using RealTime-qPCR (results not shown). [score:3]
Using primary liver tissues as another example, we detected a 29% reduction in miR-199a-3p expression in the presence of 3′UTR (Figure 5b). [score:3]
These results are summarized in Figure 6, and depict schematically that overexpression of versican 3′UTR driven by a CMV promoter could result in binding of miR-144, miR-199a-3p, and miR-136. [score:3]
There was a 39% reduction in miR-199a-3p expression in lung tissues but no significant changes were found between the transgenic and wildtype kidney tissues (Figure 5a). [score:3]
Expression of miR-199a-3p was analyzed in the primary lung and kidney of VerUTR transgenic and wildtype mice. [score:3]
We have chosen to focus on the expression of miR-199a-3p and miR-136 because miR-144 is an erythroid lineage-specific miRNA [33]. [score:3]
Rb1 is targeted by miR199a-3p and miR-144. [score:3]
In our previous studies, miR-199a-3p was found to regulate two extracellular matrix proteins, versican and fibronectin [24], and it has also been shown to be important during development [34]. [score:3]
The potential binding site of miR-144 on Rb1 3′UTR (nucleotide 674-695) was found to be close to the miR-199a-3p target site. [score:3]
Notably, the levels of miR-199a-3p and miR-136 were consistently reduced in lung tissues but not kidney tissues. [score:1]
U343 cells were co -transfected with miR-199a-3p and the luciferase constructs. [score:1]
Transient or stable transfection of 3′UTR into breast cancer cells both reduced the cellular levels of miR-199a-3p, suggesting that the degradation process is immediate and can be prolonged. [score:1]
The potential binding sites of miR-199a-3p were found on Rb1 3′UTR (nucleotide 654-675, GeneBank Accession No. [score:1]
Two additional binding sites recognized by miR-199a-3p are found in the mouse Rb1 3′UTR. [score:1]
These results suggest that the function of miR-199a-3p was likely impaired (SI Figure S3). [score:1]
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In LX-2 cells treated with TGFβ, the expression levels of miR-199a and miR-199a* were significantly higher than in untreated cells; the expression levels of miR-200a and miR-200b were significantly lower than in untreated cells. [score:5]
In this report and prior mouse studies and the expression pattern of 3 miRNAs (miR-199a-5p, 199b*, 125-5p) was found to be similar while the expression pattern of 11 miRNAs (miR-223, 221, 24, 877, 29b, 29a, 29c, 30c, 365, 148a, and 193) was partially consistent with fibrosis grade [16]. [score:5]
B. The expression levels of 3 fibrosis related genes in LX2 cells with overexpressing miR-199a, 199a*, 200a, or 200b, respectively were significantly higher than that in cells transfected with control miRNA (p<0.05; two-tailed Student t-test). [score:5]
The expression level of these 4 miRNAs was significantly different between F0 and F3 and spearman correlation analysis also showed that the expressions of these miRNAs were strongly and positively correlated with fibrosis grade (n = 105, r = 0.498(miR-199a), 0.607(miR-199a*), 0.639(miR-200a), 0.618(miR-200b), p-values<0.0001) (Figure 3). [score:5]
TGFβ -induced factor (TGIF) and SMAD specific E3 ubiquitin protein ligase 2 (SMURF2), both of which play roles in the TGFβ signaling pathway, are candidate targets of miR-199a* and miR-200b, respectively, as determined by the Targetscan algorithm. [score:5]
The relationship between expression level of miR-199 and 200 families and expression level of three fibrosis related genes. [score:5]
The expression of miR-199a* was silenced in several proliferating cell lines excluding fibroblasts [21]. [score:3]
Figure S2Comparison of the expression level of miR-199 and 200 familes in several cell lines and human liver tissue. [score:3]
Endogenous expression level of miR-199a, 199a*, 200a, and 200b in normal liver and LX2 cell as determined by microarray analysis (Agilent Technologies). [score:3]
In both the mouse and human studies, the expression levels of miR-199a, 199a*, 200a, and 200b were positively and significantly correlated to the progressed liver fibrosis. [score:3]
Lab Invest 21 Kim S Lee UJ Kim MN Lee EJ Kim JY 2008 MicroRNA miR-199a* regulates the MET proto-oncogene and the downstream extracellular signal-regulated kinase 2 (ERK2). [score:3]
Over expression of miR-199a, 199a*, 200a, and 200b was associated with the progression of liver fibrosis. [score:3]
We identified that 4 highly expressed miRNAs (miR-199a, miR-199a*, miR-200a, and miR-200b) that were significantly associated with the progression of liver fibrosis both human and mouse. [score:3]
The expression level of miR-199 and 200 families in human liver biopsy specimen by real-time qPCR. [score:3]
Furthermore, overexpression of miR-199a, miR-199a*, miR-200a and miR-200b in LX-2 cells resulted significant induction of above fibrosis-related genes compared with control miRNA (Figure 4B). [score:2]
miR-199a* is also one of the negative regulators of the HCV replication [22]. [score:2]
Down regulation of miR-199a, miR-199a* and 200a in chronic liver injury tissue was associated with the hepatocarcinogenesis [9]. [score:2]
0016081.g003 Figure 3 validation of the 4 miRNAs (miR-199a, miR-199a*, miR-200a, and miR-200b). [score:1]
validation of the 4 miRNAs (miR-199a, miR-199a*, miR-200a, and miR-200b). [score:1]
The sequences of mmu-miR-199a-5p, mmu-miR-199b, mmu-miR-199b, mmu-miR-200a, and mmu-miR-200b in mouse miRNA corresponded to the sequences of hsa-miR-199a, hsa-miR-199a*, hsa-miR-199a, hsa-miR-200a, and hsa-miR-200b in human miRNA, respectively (Table S3). [score:1]
Thus, our in vitro analysis suggested a possible involvement of miR-199a, 199a*, 200a, and 200b in the progression of liver fibrosis. [score:1]
For detection of the miRNA level by real-time qPCR, TaqMan® microRNA assay (Applied Biosystems) was used to quantify the relative expression level of miR-199a (assay ID. [score:1]
The miR-199 and miR-200 families have are circumstantially related to liver fibrosis. [score:1]
The 4 human miRNAs (miR-199a, miR-199a*, miR-200a, and miR-200b) with the largest difference in fold change between the F1 and F3 groups were chosen to validate the microarray results using stem-loop based real-time qPCR. [score:1]
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Other miRNAs from this paper: mmu-mir-199a-1
This suggests that miR-199a* repressed fibronectin expression by targeting the 3′UTR of fibronectin. [score:5]
This suggests that miR-199a* repressed versican expression by targeting the 3′UTR. [score:5]
Overexpression of versican 3′UTR would attract endogenous miR-199a* freeing versican mRNA and fibronectin mRNA for translation. [score:5]
To confirm if versican was a target of miR-199a*, U343 cells were co -transfected with versican 3′UTR-luciferase construct (lu-VUTR) or the mutant lu-Ver-mut, in which the miR-199a* target site was mutated by nucleotide replacement (Fig 4i, upper). [score:5]
To confirm that fibronectin was a target of miR-199a*, we cloned the fragment of fibronectin 3′UTR containing the miR-199a* target site. [score:5]
Thus, we hypothesized that the overexpression of versican 3′UTR would attract endogenous miR-199a*, freeing versican and fibronectin mRNA for translation. [score:5]
Mutation of the miR-199a* target site partially rescued luciferase activities. [score:4]
The potential target site of miR-199a* was mutated producing lu-FNmut. [score:3]
Luciferase activity assays showed that while presence of the versican 3′UTR reduced luciferase activities, mutation of the miR-199a* target site partially rescued luciferase activities (Fig 4i, lower). [score:3]
org), we found a great number of genes that are potentially targets of miR-199a* including versican (Genbank access number NM_004385) and fibronectin variant 1-6 (Genbank access number NM_212482, NM_212475, NM_002026, NM_212478, NM_212476, and NM_212474). [score:3]
Increase rations of versican 3′UTR bound more endogenous miR199a* and thus freeing the translation of luciferase protein, resulting in higher levels of luciferase activities (e). [score:3]
Increase amounts of versican 3′UTR bound more endogenous miR199a* and freeing the translation of luciferase protein, resulting in higher levels of luciferase activities. [score:3]
Increased ratios of versican 3′UTR bound more endogenous miR199a* and thus freeing the translation of luciferase protein, resulting in higher levels of luciferase activities (f). [score:3]
We found that the expression of the versican 3′UTR induced cell, tissue, and organ adhesion by arresting miR-199a* functions. [score:3]
Luciferase activity assays indicated that the miR-199a construct repressed luciferase activities when it harbored the versican 3′UTR, which was abolished when the potential miR-199a* target site was mutated. [score:2]
Luciferase activity assays indicated that the miR-199a construct repressed luciferase activities when it harbored the fibronectin 3′UTR, which was abolished when the potential miR-199a* target site was mutated. [score:2]
Lower, U343 cells were co -transfected with the miR-199a construct and the luciferase reporter construct harboring the versican 3′UTR (lu-VUTR) or the mutant construct (lu-Ver-mut). [score:1]
One of the positive miRNAs that showed interaction with the versican 3′UTR is miR-199a*. [score:1]
Lower, U343 cells were co -transfected with the miR-199a construct and the luciferase reporter construct harboring the fibronectin 3′UTR (lu-FNUTR) or the mutant construct (lu-FNmut). [score:1]
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[+] score: 53
Other miRNAs from this paper: hsa-mir-16-1, hsa-mir-17, hsa-mir-20a, hsa-mir-21, hsa-mir-23a, hsa-mir-100, hsa-mir-103a-2, hsa-mir-103a-1, hsa-mir-107, hsa-mir-16-2, mmu-mir-1a-1, mmu-mir-23b, mmu-mir-125b-2, mmu-mir-130a, mmu-mir-9-2, mmu-mir-145a, mmu-mir-181a-2, mmu-mir-184, mmu-mir-199a-1, hsa-mir-199a-1, mmu-mir-205, mmu-mir-206, hsa-mir-181a-2, hsa-mir-181b-1, hsa-mir-199a-2, hsa-mir-205, hsa-mir-181a-1, hsa-mir-214, hsa-mir-219a-1, hsa-mir-223, mmu-mir-302a, hsa-mir-1-2, hsa-mir-23b, hsa-mir-125b-1, hsa-mir-130a, hsa-mir-145, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-125b-2, hsa-mir-184, hsa-mir-206, mmu-mir-16-1, mmu-mir-16-2, mmu-mir-20a, mmu-mir-21a, mmu-mir-23a, mmu-mir-103-1, mmu-mir-103-2, rno-mir-338, mmu-mir-338, rno-mir-20a, hsa-mir-1-1, mmu-mir-1a-2, hsa-mir-181b-2, mmu-mir-107, mmu-mir-17, mmu-mir-100, mmu-mir-181a-1, mmu-mir-214, mmu-mir-219a-1, mmu-mir-223, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-181b-1, mmu-mir-125b-1, hsa-mir-302a, hsa-mir-219a-2, mmu-mir-219a-2, hsa-mir-302b, hsa-mir-302c, hsa-mir-302d, hsa-mir-367, hsa-mir-372, hsa-mir-338, mmu-mir-181b-2, rno-mir-9a-1, rno-mir-9a-3, rno-mir-9a-2, rno-mir-16, rno-mir-17-1, rno-mir-21, rno-mir-23a, rno-mir-23b, rno-mir-100, rno-mir-103-2, rno-mir-103-1, rno-mir-107, rno-mir-125b-1, rno-mir-125b-2, rno-mir-130a, rno-mir-145, rno-mir-181a-2, rno-mir-181b-1, rno-mir-181b-2, rno-mir-184, rno-mir-199a, rno-mir-205, rno-mir-206, rno-mir-181a-1, rno-mir-214, rno-mir-219a-1, rno-mir-219a-2, rno-mir-223, hsa-mir-512-1, hsa-mir-512-2, rno-mir-1, mmu-mir-367, mmu-mir-302b, mmu-mir-302c, mmu-mir-302d, rno-mir-17-2, hsa-mir-1183, mmu-mir-1b, hsa-mir-302e, hsa-mir-302f, hsa-mir-103b-1, hsa-mir-103b-2, rno-mir-9b-3, rno-mir-9b-1, rno-mir-9b-2, rno-mir-219b, hsa-mir-23c, hsa-mir-219b, mmu-mir-145b, mmu-mir-21b, mmu-mir-21c, mmu-mir-219b, mmu-mir-219c, mmu-mir-9b-2, mmu-mir-9b-1, mmu-mir-9b-3
This miRNA expression pattern is in accordance with published MRF expression results, potentially suggesting that changes in miR-199a-5p expression may be concomitant with the initiation of MBP expression. [score:9]
Besides having a strong target bias for C11Orf9, miR-199a-5p also has a significant bias for targetting DDR1, a tyrosine kinase receptor found to be upregulated during in vivo remyelination and in vitro oligodendrocyte differentiation [34]. [score:8]
The highly differential expression of miR-199a-5p, along with its high number of stringent evolutionarily conserved target sites to DDR1 mRNA, suggests a likely regulatory interaction involved in oligodendrocyte differentiation. [score:6]
From our top ten differentially expressed miRNAs at the early OP to mid OP stage transition, two miRNAs (miR-199a-5p and miR-145) show strong target bias towards C11Orf9. [score:5]
Interestingly, miR-199a-5p demonstrated a large decrease in expression beginning at the early OP stage and an increase at the final stage. [score:3]
However, while the increase of miR-199a-5p at the final stage corresponds with the decreased MRF expression at this transition, additional validation is necessary. [score:3]
Target predictions for miR-199a-5p showed high likelihood for C11Orf9 repressive interactions. [score:3]
Similarly, the expression pattern of miR-145 at these stages conformed to the same trend as miR-199a-5p. [score:3]
For instance, miR-145 and miR-199a-5p within our data showed similar expression patterns throughout differentiation and both contain conserved 8mer predicted seed pairings to multiple sites within the 3′-UTR of C11orf9. [score:3]
Importantly, miR-199a-5p showed a sharp ∼8.6-fold decrease in expression from the early OP to mid OP stage, followed by minimal ∼1.1-fold change during the mid OP to late OP stage transition. [score:3]
MiR-199a-5p, with a ∼8.6-fold decrease from OP1 to OP2 stage, contains four total evolutionarily conserved target sites (three 8mer, one 7mer-1A) within the 3′ UTR of DDR1 mRNA. [score:2]
Therefore, these data suggest that miR-199a-5p and miR-145 may be simultaneously regulating the human homolog of MRF. [score:2]
MiR-199a-5p has three sites with evolutionarily conserved 8mer seed matches to the 3′ UTR of C11Orf9, suggesting a high probability for miRNA-mRNA interactions. [score:1]
Then, miR-199a-5p levels rose (∼1.7 fold) at the final stage of differentiation. [score:1]
The key miRNAs discussed in this manuscript were validated by conducting real-time qRT-PCR for samples from the appropriate stages, including the following: miR-199a and miR-145 at the OP1, OP2, OP3, and OL stages; miR-214 at the OP1 and OP2 stages; miR-184 and miR-1183 at the GP and OP1 stages (Table 1 ). [score:1]
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For instance, hepatic stellate cells had been found to express miR-199, which influenced stellate cell activity by regulating collagen synthesis and represented a general mechanism contributing to hepatic fibrosis (Lino et al., 2013); the liver sinusoidal endothelial cells also express the miR-199 and this miRNA might serve as a negative regulator of the function of liver sinusoidal endothelial cells in some chemical exposures, such as alcohol (Wang et al., 2012); the increased miR-21 expression was detected in the hepatic stellate cells with the treatment of ethanol and interleukin-6 (Francis et al., 2014). [score:9]
It was demonstrated that the expression of miR-21a-3p was upregulated and miR-199a-5p and miR-139-5p were downregulated in FMT water extract -treated group when compared with control group (Figure 5), which was consistent with the results from microarray analysis. [score:8]
miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta -induced lung fibroblast activation by targeting caveolin-1. PLoS Genet. [score:6]
Specifically, the increase of miR-21a expression was detected in alcoholic liver injury (Francis et al., 2014), and the expression of miR-199a-5p was elevated in both bile acid- and thapsigargin-stimulated cultured hepatocytes, as well as in the liver of bile duct-ligated mice (Dai et al., 2013). [score:5]
Among these four miRNAs, the miR-199a-5p and miRNA-21 were found to be expressed by other cell types of liver and contributed to liver diseases. [score:5]
Moreover, the target genes of four miRNAs could be predicted by target filter analysis in IPA database, including miR-139-5p, miR-199a-5p, miR-2861, and miR-3960. [score:5]
Thus, the downregulation of miR-139-5p and miR-199a-5p in this study may indicate the potential risk of hepatic carcinoma caused by FMT water extract. [score:4]
We noticed that ASK1, PKC, and JUN affected by the differentially expressed miRNAs of FMT water extract, i. e., miR-139-5p and miR-199-5p. [score:3]
Moreover, the dysregulation of miR-21a and miR-199a-5p have been also reported by other liver injury mo dels. [score:2]
It was regulated by miR-139-5p and miR-199a-5p and activated by the oxidative stress (Lee et al., 2001). [score:2]
Similarly, miR-199a-5p, one of the most abundant miRNAs in hepatocytes, prevented hepatocyte apoptosis (Dai et al., 2013). [score:1]
microRNA-199a-5p protects hepatocytes from bile acid -induced sustained endoplasmic reticulum stress. [score:1]
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Candidate target genes regulated by the upregulated miRNAs (mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p and mmu-miR-16-1-3p). [score:7]
Of these miRNAs, four were upregulated (mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p, and mmu-miR-16-1-3p), and one was downregulated (mmu-miR-212-3p). [score:7]
Among the upregulated miRNAs, 292, 178, 164, and 243 candidate target genes were predicted for mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p, and mmu-miR-16-1-3p, respectively (S2 Table). [score:6]
Mmu-miR-199a-5p is upregulated during the fibrogenic response to tissue injury and mediates TGFbeta -induced lung fibroblast activation by targeting caveolin-1 [31]. [score:6]
Of the five differentially expressed miRNAs, four miRNAs, namely, mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p, and mmu-miR-16-1-3p, were significantly upregulated in the vitrified blastocysts. [score:6]
Mmu-miR-199a* was found to be differentially expressed in the mouse uterus during implantation in coordination with the expression of cyclooxygenase-2 (COX-2), a gene critical for implantation [26]. [score:5]
In this study, 5 of the 760 identified miRNAs, namely, mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p, mmu-miR-16-1-3p, and mmu-miR-212-3p, showed significantly different expression between the vitrified and fresh mouse blastocysts. [score:3]
The mmu-miR-199a-5p miRNA showed the highest expression level at the embryonic stage at 12.5 days postcoitus [29]. [score:3]
A previous study reported that mmu-miR-199a-5p could silence the self-renewal of mouse embryonic stem cells [30]. [score:1]
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As miR-199a, miR-455 and miR-1192 were up-regulated in PPARγ DN animals this may indicate that PPARγ normally inhibits their expression directly (e. g. by recruiting transcriptional inhibitors to those promoters) or indirectly (by the PPARγ specific transctivation of a transcriptional inhibitor). [score:14]
Interestingly, only four of the 21 regulated miRNAs (miR-199a, miR-455, miR-592, and miR-1192) were solely dependent on the genotype; independent of diet, the expression of miR-199a, miR-455 and miR-1192 was up-regulated in PPARγ DN animals whereas miR-592 was down-regulated in these mice compared to wt. [score:9]
The expression of four miRNAs was only dependent on the PPARγ expression status of the animals; miR-199, miR-455 and miR-1192 were upregulated in mice with dominant -negative PPARγ irrespective of the diet given to the animals, the fourth, miR-592, was reduced in mutant animals (“a” in Fig. 5C). [score:8]
Among those, were miR-7a, miR-7b, miR-146b, miR-199a and miR-199b* that were regulated at very early time-points after induction of MSCs and show the same direction of up- or down-regulation in the NIH/3T3 mo del. [score:6]
Five of these eight miRNAs (miR-199a, miR-7, miR-146b, miR-7b, and miR-199b*) were similarly regulated early during the adipogenesis process of MSCs, indicating that their regulation was due to cell cycle arrest rather than adipogenesis. [score:3]
Members of 5 miRNA families were coordinately up regulated during adipogenesis, namely let-7a, let-7c, let7-e, let-7g, and let-7i; members of the miR-29 family miR-29a, miR-29b, and miR-29c; miR-30a_1, miR-30b, miR-30c, miR-30d, and miR-30e; miR-181a, and miR-181b; miR-199a, miR-199a-3p, miR199-b*. [score:2]
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Therefore, expression of select miRNAs, including the miR-199 and miR-30 families, decreases during reprogramming and may allow for the upregulation of SIRT1 protein expression. [score:8]
This correlated with the downregulation of miR-199 andmiR-30 families that target SIRT1 (Figure 6C). [score:6]
Expression of miR-199a and b is highest in skin (Supplementary Figure 3), and limiting the expression of these specific miRNAs may be a prerequisite for reprogramming of MEFs. [score:5]
As previously discussed, miR-199a and b were strongly upregulated during mESC differentiation (Figure 3). [score:4]
As predicted, reprogramming of MEFs into iPS cells was accompanied by a downregulation of miR-199a and b by 3.3-fold and 5.8-fold, respectively (Figure 6D). [score:4]
qRT-PCR analysis of miR-199a and b expression relative to miR-16 in mESCs, MEFs, and various mouse tissues. [score:3]
Supplementary Figure 3 qRT-PCR analysis of miR-199a and b expression relative to miR-16 in mESCs, MEFs, and various mouse tissues. [score:3]
miR-199 is highly expres-sed in mouse embryonic fibroblasts and skin. [score:1]
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Mir-214/mir-214* and mir-199a-5p/mir199a-3p are clustered on pri -mir-199a-2 within the Dnm3os gene in mouse as well as in human genome and were strongly upregulated by VPA (8.7/20.6- and 9.8/11.1-fold, respectively), while exposure to arsenite significantly reduced the expression of these miRNAs (−2.4/−1.97- and −2.4/−2.9-fold, respectively). [score:6]
In four independent differentiation procedures we could confirm the microarray data (Fig. 5A)–that is, a strong concentration -dependent induction of muscle-specific/abundant miRNA (mir-206, mir-10a, mir-214, mir-145, mir-143, mir-199a) and a significant downregulation of the expression of neuro-specific miRNAs (mir-124, mir-128, mir-137, mir-491, mir-383) in comparison to the solvent control. [score:6]
The mir-214/mir-199a cluster is regulated during embryonic development by transcription factor Twist1 [77], [78], the expression of which was significantly induced by VPA in our cell system (2.2-fold). [score:5]
Otx2 and Zic4 are predicted targets of mir-206, while Otx1 and Zic3 are predicted targets of mir-199a and the muscle-specific mir-133a, respectively. [score:5]
Comparing to the solvent control, in cells treated with VPA we observed a strong upregulation of myogenic miRNAs (myo- mirs: mir-206, mir-133a,b), or miRNAs shown to be involved in muscle differentiation and specification (mir-10a, mir-143/ mir-145 cluster, mir-214, mir-322, mir-199a). [score:4]
Moreover, mir-214, mir-145 and mir-199a were significantly downregulated in these cells. [score:4]
Importantly, clustering of some miRNA genes within the genome is also conserved between human and mice (e. g. mir-206/mir-133b, mir-214/mir199a, mir-10a/HoxB4, mir-145/143 clusters, all of which have been studied here) (www. [score:1]
Mir-214, mir-199a and mir-145 were also induced but not to such an extent as seen for VPA. [score:1]
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Pathways analysis predicted that miR-101a is targeting TGFBR1 and SMAD3, miR-582 is targeting TGFB2, SMAD1, miR-425 is targeting TGFBR2 and FST, miR-199a is targeting ACVR2a, miR-148 is targeting ACVR1, and miR-103 is targeting BMP2 in TGF- β signaling pathway. [score:13]
Predicted target and pathway analysis concluded that miR-101a is targeting TGFBR1 and SMAD3, miR-425 is targeting TGFBR2 and FST, miR-582 is targeting SMAD1 and TGFB2, miR-148 is targeting ACVR1, and miR-199a is targeting AcvR1a gene in the TGF- β signaling pathway. [score:13]
Six isomiRs i. e. mmu-miR-16a-2, mmu-miR-133a-1, mmu-miR-188b-2, mmu-miR-199a-2, mmu-miR-486 and mmu-miR-26b-5P were selected. [score:1]
Of the 8 known miRNAs examined, 7 miRNAs (miR-425, miR-26a, miR-1a, miR-199a, miR-101, miR-378, and miR-151) showed a consistent pattern with the deep sequencing data (Figures 6(a)– 6(j)). [score:1]
Out of 8 known miRNAs, 6 miRNAs have been functionally linked to myogenesis (i. e., miR-1a, miR-26a, miR-133a and miR-199a, miR-101, and miR-378 [38, 50, 51, 54, 55, 70]). [score:1]
In some other cases, such as mmu-miR-181b-2, mmu-miR-199a-2, and mmu-miR-16a-2, more than one highly abundant isoform was present (Figure 3), indicating that some miRNAs have more than one isoformin specific tissues. [score:1]
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Moreover, miR-199a was reported to act as an inhibitor of chondrogenesis by targeting Smad1 [17]. [score:5]
Lin Ea Kong L Bai X-H Luan Y Liu C-J miR-199a, a bone morphogenic protein 2-responsive MicroRNA, regulates chondrogenesis via direct targeting to Smad1J. [score:5]
As expected, miR-199a had a similar pattern of expression (Supplementary Fig.   S1), consistent with the fact that both miRNAs are originated from the same transcript (Dnm3os). [score:3]
In the previous section we confirmed that miR-214 and miR-199a have similar temporal expression patterns, consistent with the fact that both miRNAs derive from the same transcript. [score:3]
This is also in agreement with previous studies showing similar patterns of expression for miR-199 and miR-214 in different systems 15, 20, 21. [score:3]
Lee Y-B Twist-1 regulates the miR-199a/214 cluster during developmentNucleic Acids Res. [score:3]
Although the putative role of miR-214 in chondrogenesis remains generally unknown, it is now accepted that the Dnm3 opposite strand (Dnm3os) transcript, which encodes miR-214 and miR-199a cluster [15], is crucial for normal mouse skeletal development, including cartilage formation [16]. [score:2]
Identification and analysis of Dnm3os promoter sequencesPre-miR-199a-2 sequences were retrieved from miRbase database (http://www. [score:1]
We searched for conserved putative skeletal transcription factor binding sites (TFBS) in a 2.5 kilobase (kb) region upstream of pre-miR-199 (putative Dnm3os promoter) of human, mouse, Xenopus, medaka, stickleback, Tetraodon, fugu and zebrafish. [score:1]
The cluster miR-199a-2/miR-214 is transcribed from the opposite strand of Dynamin 3 (Dnm3), in a common primary transcript called Dnm3os 15, 16, 18. [score:1]
org) and genomic sequences with 2.5 kilobase (Kb) were collected starting immediately upstream of pre-miR-199a-2 (now on called Dnm3os promoter). [score:1]
Pre-miR-199a-2 sequences were retrieved from miRbase database (http://www. [score:1]
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From the miRNA database, we also observed that TWEAK down-regulates miRNAs that are targeting proliferation and remo deling of muscles (miR-107), matrix metalloprotease such as aggrecanase-2 (miR-192), adamtsl-3 (miR-199-3p), and genes involved in increasing cell growth and proliferation, and microtubule -associated proteins (miR-23b) (Table 3). [score:6]
Some of the important miRs with known/putative targets and differentially regulated by TWEAK are presented in Figure 3. Our results showed that TWEAK reduced the expression of muscle-specific miR-1, miR-133a, miR-133b and miR-206 in addition to several other miRs including miR-27, miR-23, miR-93, miR-199, miR-107, and miR-192 (Figure 3A). [score:6]
miR-148b −2.6183 decrease apoptosis miR-152 −2.3341 Increase cell growth miR-17 −6.2545 Bcl2, N-myc miR-181c −3.5756 proliferation and remo deling of muscles miR-190b −2.2 Binds to Ubiquitin-specific protease 46, increase cell growth miR-192 −2.4871 Increase cell growth miR-199a-3p −1.9 Activin receptor IIA, Map3k4 miR-218-1 −2.2887 Increase cell growth miR-23b −2.1623 Increase Cell growth, proliferation miR-26a −2.4565 decrease proapoptotic signaling miR-27a −2.7 Ubiquitin-conjugating enzyme E2N miR-27b −3 Ubiquitin-conjugating enzyme E2N miR-296-3p −7.3378 Increase cell growth, decrease apoptosis miR-322 8.7 Hydroxysteroid (17-beta) dehydrogenase 7 miR-455 129.249 Up-regulated brown adipocyte differentiation miR-470 3.2 TGFB -induced factor homeobox 1 miR-715 18.25 Fucosyltransferase 1 miR-7a −6.2174 Increase cell growth, decrease apoptosis miR-93 −48.423 Map3k14 (NIK) miR-98 1.8 Tripartite motif-containing 6, insulin-like growth factor 2 mRNA binding protein 1 In order to understand the interaction between different genes, we generated common networks using Ingenuity Pathway Analysis (IPA) software. [score:4]
In addition to MyomiRs, TWEAK also down-regulated a few more miRNAs such as miR-27a and b, miR-93, miR-199a-3p, miR-107, miR-192, and miR-23b (Fig. 5A). [score:4]
Low-density miRNA array of TWEAK -treated C2C12 myotubes showed down-regulation of miR-1, miR-133a, miR-133b, miR-206, miR-27, miR-23, miR-93, miR-199, miR-107, and miR-192. [score:4]
miR-148b −2.6183 decrease apoptosis miR-152 −2.3341 Increase cell growth miR-17 −6.2545 Bcl2, N-myc miR-181c −3.5756 proliferation and remo deling of muscles miR-190b −2.2 Binds to Ubiquitin-specific protease 46, increase cell growth miR-192 −2.4871 Increase cell growth miR-199a-3p −1.9 Activin receptor IIA, Map3k4 miR-218-1 −2.2887 Increase cell growth miR-23b −2.1623 Increase Cell growth, proliferation miR-26a −2.4565 decrease proapoptotic signaling miR-27a −2.7 Ubiquitin-conjugating enzyme E2N miR-27b −3 Ubiquitin-conjugating enzyme E2N miR-296-3p −7.3378 Increase cell growth, decrease apoptosis miR-322 8.7 Hydroxysteroid (17-beta) dehydrogenase 7 miR-455 129.249 Up-regulated brown adipocyte differentiation miR-470 3.2 TGFB -induced factor homeobox 1 miR-715 18.25 Fucosyltransferase 1 miR-7a −6.2174 Increase cell growth, decrease apoptosis miR-93 −48.423 Map3k14 (NIK) miR-98 1.8 Tripartite motif-containing 6, insulin-like growth factor 2 mRNA binding protein 1 A) C2C12 myotubes were treated with 10ng/ml of TWEAK for 18h following isolation of total RNA enriched with small RNAs. [score:4]
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The expression patterns of miR-199a, miR-470, miR-871, shows relatively higher expression in small preantral follicles as compared to large antral follicles with the similar expression pattern as the results of deep sequencing. [score:6]
Others predominantly expressed miRNAs e. g., mmu-mir-125b, mmu-mir-199a-3p, mmu-mir-29a and mmu-mir-15b targets several ovarian genes and involved in several biological functions like cell signaling, cell death, cell cycle regulation, cellular growth and differentiation and endocrine system [37]. [score:6]
Likewise, mmu-mir-21, mmu-mir-125b, mmu-mir-16b, mmu-mir-143 and mmu-mir-199a-3p were expressed abundantly in all libraries despite of changes in expression with development thus suggesting its role in basic reproductive activities. [score:6]
a) 6d-8d, b) 8d-12d, c) 12d-15d, d) 15d-21d, e) 21d-P6, f) P48-h6 To further validate these differentially expressed miRNAs identified from the mouse ovary, the expression levels of miR-199a, miR-470, miR-871, miR-34c let-7a, miR-7a, miR-351, miR-191 were further examined in different size follicles (i. e., 100 μm −130 μm, 200 μm -280 μm, 450 μm -550 μm, 500 μm -600 μm) using qRT-PCR assay. [score:4]
A) Expression profile of mmu-mir-199a in sequencing data. [score:3]
a) Expression profile of miR-199a through qRT-PCR. [score:3]
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Analysis of global profiles of miRNA expression in skeletal muscle with microarray shows that expression of 4 miRNAs (miR-29a, miR-29b, miR-29c and miR-150) are up-regulated [23], whereas expression of 11 miRNAs (miR-379, miR-127, miR299-5p, miR-434-3p, miR-335, miR130a, miR-19b, miR-451, miR-148a, miR-199a and miR-152) are down-regulated in skeletal muscle of type 2 diabetic rats [23]. [score:13]
For example, it has been shown that expression of 4 miRNAs (miR-29a, miR-29b, miR-29c and miR-150) is up-regulated [23], whereas expression of 11 miRNAs (miR-379, miR-127, miR299-5p, miR-434-3p, miR-335, miR130a, miR-19b, miR-451, miR-148a, miR-199a and miR-152) is down-regulated in skeletal muscle of type 2 diabetic rats [23]. [score:11]
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Here, we found that the expression levels of miR-199 family members were up-regulated (3–4 fold) in the liver of S. japonicum-infected mice at 45 dpi compared with that of uninfected mice, indicating that dysregulation of miR-199 members may represent a general mechanism contributing to the fibrotic process. [score:6]
MiRNAs significantly dysregulated in the mid-phase of infection (dpi 30), such as mmu-miR-146b and mmu-miR-155, may relate to the regulation of hepatic inflammatory responses, whereas miRNAs exhibiting a peak expression in the late phase of infection (dpi 45), such as mmu-miR-223, mmu-miR-146a/b, mmu-miR-155, mmu-miR-34c, mmu-miR-199, and mmu-miR-134, may represent a molecular signature of the development of schistosomal hepatopathy. [score:6]
Further, Murakami et al. observed that the progression of liver fibrosis was related with over -expression of the miR-199 and 200 families in a CCL4 -induced murine mo del [41], and miR-199a-5p was also observed to mediate TGF-β -induced lung fibroblast activation by targeting Caveolin-1 [42]. [score:5]
In addition, some other miRNAs previously reported to be associated with fibrosis, such as miR-34c, miR-199, and miR-214, also exhibited a peak expression in the liver of infected mice at 45 dpi (Table 2 and Figure 3). [score:3]
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Recently, He et al. found that klotho was a target gene of miR-199a-5p in cancer 18, suggesting that the miR-199 family may be involved in the regulation of klotho in DN. [score:4]
A previous study reported that serum miR-199a was up-regulated in diabetic mice and that this resulted in an increase in beta cell apoptosis 38. [score:4]
Combined with the decreased antioxidant abilities levels in the cells exposed to 20 mmol/L glucose, we hypothesized that high glucose leads to excessive production of reactive oxygen species and depletion of antioxidant enzymes, resulting in oxidative stress, up-regulation of histone acetylation, and activation of miR-199a-5p. [score:4]
Real-time PCR was used to detect serum and renal tubular epithelial cell miR-199-5p expression (D). [score:3]
Another study of T2DM patients found that plasma miR-199a was overexpressed and that it played a role in repressing glucose uptake 39. [score:3]
To investigate the effects of miR-199b-5p and klotho on renal function in vivo, the STZ -induced diabetic mice were injected with AAV-miR-199b-5p to overexpress miR-199-5b. [score:1]
The effects of miR-199b-5p and klotho on renal function in vivoTo investigate the effects of miR-199b-5p and klotho on renal function in vivo, the STZ -induced diabetic mice were injected with AAV-miR-199b-5p to overexpress miR-199-5b. [score:1]
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Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-16-1, hsa-mir-21, hsa-mir-16-2, mmu-let-7g, mmu-let-7i, mmu-mir-9-2, mmu-mir-151, mmu-mir-10b, hsa-mir-192, mmu-mir-194-1, mmu-mir-199a-1, hsa-mir-199a-1, mmu-mir-122, hsa-mir-10a, hsa-mir-10b, hsa-mir-199a-2, hsa-mir-199b, hsa-mir-210, hsa-mir-214, mmu-let-7d, hsa-let-7g, hsa-let-7i, hsa-mir-122, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-194-1, mmu-mir-192, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-16-1, mmu-mir-16-2, mmu-mir-21a, mmu-mir-10a, mmu-mir-210, mmu-mir-214, mmu-mir-199b, mmu-mir-9-1, mmu-mir-9-3, hsa-mir-194-2, mmu-mir-194-2, hsa-mir-365a, mmu-mir-365-1, hsa-mir-365b, hsa-mir-151a, gga-let-7i, gga-let-7a-3, gga-let-7b, gga-let-7c, gga-mir-16-1, gga-mir-194, gga-mir-10b, gga-mir-199-2, gga-mir-16-2, gga-let-7g, gga-let-7d, gga-let-7f, gga-let-7a-1, gga-mir-199-1, gga-let-7a-2, gga-let-7j, gga-let-7k, gga-mir-122-1, gga-mir-122-2, gga-mir-9-2, mmu-mir-365-2, gga-mir-9-1, gga-mir-365-1, gga-mir-365-2, hsa-mir-151b, mmu-mir-744, gga-mir-21, hsa-mir-744, gga-mir-199b, gga-mir-122b, gga-mir-10a, gga-mir-16c, gga-mir-214, sma-let-7, sma-mir-71a, sma-bantam, sma-mir-10, sma-mir-2a, sma-mir-3479, sma-mir-71b, mmu-mir-21b, mmu-let-7j, mmu-mir-21c, mmu-let-7k, gga-mir-365b, sma-mir-8437, sma-mir-2162, gga-mir-9-3, gga-mir-210a, gga-mir-9-4, mmu-mir-9b-2, mmu-mir-9b-1, mmu-mir-9b-3, gga-mir-9b-1, gga-mir-10c, gga-mir-210b, gga-let-7l-1, gga-let-7l-2, gga-mir-122b-1, gga-mir-9b-2, gga-mir-122b-2
In contrast, the miRNAs up-regulated in the liver (miR-199-3p, miR-199-5p, miR-21, miR-214 and miR-210) showed significantly higher levels in mouse serum at 12 weeks post infection (Fig. 2), however these failed to differentiate S. mansoni infected from uninfected humans (Fig. S4). [score:4]
Temporal expression analysis of miR-199, miR-214, miR-21, miR-210, miR-122, miR-192 and miR-194 in the liver during S. mansoni infectionBetween weeks 6 and 12, female parasites continue to produce ∼300 eggs per day [51], resulting in an increase in the number of granulomas in the liver and the development of fibrosis [45]. [score:4]
The miRNAs that displayed the largest differential expression included miR-199a and miR-214, which are known to be altered in liver fibrosis caused by hepatitis C infection or induced by carbon tetrachloride [47], [48]. [score:3]
Temporal expression analysis of miR-199, miR-214, miR-21, miR-210, miR-122, miR-192 and miR-194 in the liver during S. mansoni infection. [score:3]
Thirty-three mouse miRNAs were differentially expressed in infected compared to naïve mice (>2 fold change, p<0.05) including miR-199a-3p, miR-199a-5p, miR-214 and miR-21, which have previously been associated with liver fibrosis in other settings. [score:2]
Consistent with the array results, there was an increase in miR-199-5p, miR-199-3p, miR-214, miR-21, miR-210, and a reduction of miR-192, miR-194, miR-365, miR-122 and miR-151 in the liver tissue of S. mansoni infected mice as compared to naïve mice; miR-9 and miR-744 did not display differential expression and were not analysed further (Table 1). [score:2]
The 5 host miRNAs were detectable in serum (miR-21, miR-199-3p, miR-199-5p, miR-210, miR-214) but showed variable abundance and failed to differentiate ‘egg -positive’ and ‘egg -negative’ participants (Fig. S4). [score:1]
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Other miRNAs from this paper: mmu-mir-101a, mmu-mir-199a-1, mmu-mir-200a, mmu-mir-96
The heat scale at the right of the panel represents a linear scale, with magenta, black and turquoise representing high, average and low expression, respectively; Expression profiles of miR-96-5p (b), miR-101a-3p (c), miR-199a-5p (d) and miR-200a-3p (e) over 24 h in mesencephalon examined by a microarray or qRT-PCR. [score:5]
These oscillations of miRNAs such as miR-96-5p, miR-199a-5p and miR-200a-3p are unique in mature miRNA processing; other miRNAs such as miR-101a-3p exhibit no significant diurnal changes in expression (Supplementary Fig. 8). [score:3]
Significant rhythmicity of the expression patterns of miR-96-5p and miR-199a-5p miR-200a-3p was revealed (P=0.0082, 0.026 and 0.023, respectively) but not of miR-101a-3p (P=0.053). [score:3]
Among the candidates with diurnal oscillations, our computational analysis of candidate miRNA prediction using established programmes revealed three miRNAs-miR-96-5p, miR-199a-5p and miR-200a-3p-as possible candidates, with oscillations that target the 3′-UTR of human, mouse and rat EAAC1. [score:3]
The transfection of miR-96-5p or miR-101a-3p into HEK293 cells decreased EAAC1 protein compared with control miRNA (Steel’s test; P<0.05), whereas miR-199a-5p and miR-200a-3p had no effects on protein expression (Fig. 4a,b). [score:2]
Using qRT–PCR, we confirmed that miR-96-5p, miR-199a-5p and miR-200a-3p oscillate in a diurnal manner as shown by the microarray data (Cosinor; P=0.0082, 0.026 and 0.023, respectively) but that miR-101a-3p does not (Cosinor; P=0.053). [score:1]
MiR-199a-5p and miR-200a-3p had especially large amplitudes, with more than threefold changes and peaks at ZT5 and ZT14, respectively (Fig. 3d,e). [score:1]
In contrast, miR-199a-5p and miR-200a-3p had no effect on the luciferase activity, which is also consistent with the western blotting results (Fig. 4a,d, Supplementary Fig. 9b). [score:1]
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For example, Hlf was targeted by mmu-miR-18b-5p, mmu-miR-429-3p and mmu-miR-291a-3p; Cnot6 and Zfp91 were targeted by mmu-miR-206-3p and mmu-miR-291a-3p; Rbfox2 was targeted by mmu-miR-429-3p and mmu-miR-449c-5p; Aebp2 was targeted by mmu-miR-125a-3p, mmu-miR-223-3p and mmu-miR-496-3p; Nfya was targeted by mmu-miR-199a-5p, mmu-miR-216b-5p and mmu-miR-671-5p; Nucks1 was targeted by mmu-miR-142-3p, mmu-miR-146a-5p and mmu-miR-223-3p; Notch2 was targeted by mmu-miR-146a-5p and indirectly via Ascl1 by mmu-miR-1903. [score:16]
MP1, MP2, MP3, MP4, MP5, MP6 and MP7 had a common core comprised of mmu-miR-1-3p, mmu-miR-145-5p, mmu-miR-18a-5p, mmu-miR-199a-5p, mmu-miR-200b-3p, mmu-miR-223-3p, mmu-miR-291a-3p, mmu-miR-34a-5p and their target mRNAs. [score:3]
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miR-199a that was upregulated in chondroblasts was found to target HIF1α miR-124a was also upregulated in chondroblasts with RFX1 as its target. [score:11]
HIF1α was predicted to be regulated by miR-199a that was over 12-fold upregulated in chondroblasts. [score:5]
Hong et al. (2005) Science 309:1074–1078 miR-199a HIF1α SOX9, HDAC4, RUNX2, ARNT Regulation of cell differentiation and proliferation in hypoxia. [score:2]
When the existence of genomic miRNA clusters were analysed for the miRNAs studied here, two miRNAs were found to be located in clusters, namely miR-199a /miR-214 and miR-96 /miR-182/miR-183. [score:1]
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We previously suggested that of the miRs analyzed, nine (miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805 and miR-194) stand out as being differentially expressed in C57BL/6 mice undergoing IRI compared to the expression observed in mice undergoing a sham procedure [14]. [score:4]
Movie showing the rotation of a three-dimensional plot of the first three PCs obtained by performing PCA on all expression data obtained for kidneys from C57BL/6 mice following IRI (blue line) or sham surgery (red lines) in which we eliminated miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805 and miR-194 from the analysis. [score:3]
Our previous work showed that in C57BL/6 mice, expression of miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805 and miR-194 is significantly different between IRI and sham control groups at all times analyzed [14]. [score:3]
Panel C, Shown is a three-dimensional plot of the first three PCs obtained by performing PCA on all expression data obtained for kidneys from C57BL/6 mice following IRI (blue line) or sham surgery (red lines) in which we eliminated miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805 and miR-194 from the analysis. [score:3]
Shown is a three-dimensional plot of the first three PCs obtained by performing PCA on expression data for miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805 and miR-194 obtained for kidneys from C57BL/6 mice following IRI (blue line) or sham surgery (red lines) (Movie S3). [score:3]
Shown is a three-dimensional plot of the first three PCs obtained by performing PCA on expression data for miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805 and miR-194 obtained for kidneys from C57BL/6 mice following IRI (blue line) or sham surgery (red lines). [score:3]
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[+] score: 18
For miRNAs which suppress HBV replication, such as miR-210, miR-199a-3p (47), miR-125a-5p (48), miR-29c (49), miR-122 (50, 51) and miR-141 (52), besides the three which directly target HBV transcripts, most act through targeting a positive regulator of HBV, such as miR-141, which targets peroxisome proliferator-activated receptor alpha (PPARA). [score:11]
As previously noted (31), three miRNAs (miR-210, miR-199a-3p and miR-125a-5p) have been reported to interact directly with HBV transcripts, while the other miRNAs indirectly regulate HBV life cycles by influencing relevant cellular proteins. [score:4]
Zhang G. L. Li Y. X. Zheng S. Q. Liu M. Li X. Tang H. Suppression of hepatitis B virus replication by microRNA-199a-3p and microRNA-210Antiviral Res. [score:3]
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[+] score: 17
IPost up-regulated miR-1, miR-15b, miR-21, miR-24, miR-26a, miR-27, miR-133a, miR-199a, miR-214, miR-208 and miR-499, while down-regulated miR-23a and miR-9 as compared with Sham group. [score:6]
Compared with sham group, the expressions of miR-1, miR-15b, miR-21, miR-24, miR-26a, miR-27, miR-133a, miR-199a, miR-214, miR-208 and miR-499 were increased in IPost hearts, while miR-9 and miR-23a were down-regulated in IPost mo dels. [score:5]
As previously reported, a collection of miRNAs were abnormally expressed in ischemic mouse hearts in response to I/R injury, such as miR-1, miR-9, miR-15b, miR-21, miR-23a, miR-24, miR-26a, miR-27, miR-133a, miR-199a, miR-208, miR-214 and miR-499 [20, 21, 28]. [score:3]
Then real-time quantitative PCR was performed to quantify the expression level of miR-1, miR-9, miR-15b, miR-21, miR-23a, miR-24, miR-26a, miR-27, miR-133a, miR-199a, miR-208, miR-214 and miR-499 with SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. [score:3]
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[+] score: 17
We focused on miR-199b-5p due to its ability to decrease HES1 expression under transient over -expression, as compared to miR-199a-5p (Text S1, Fig. S1A, B). [score:4]
Recently it has also been shown that miR-199a*, which has the same sequence as miR-199b-5p, is involved in caspase activation by potentially acting through Met proto-oncogene down-regulation [42]. [score:4]
Figure S1 A) Representative Western blots from transient transfection of HEK293 cells with expression constructs for miR-199b-5p and miR-199a. [score:3]
MiR-199b-5p over -expression lowered the levels of the endogenous HES1 protein; in contrast, miR-199a induced an increase in HES1 levels 48 h after transfection. [score:3]
MiR-199b-5p and miR-199a-5p were the better scoring miRNAs, and were predicted to bind the 3′UTR of human bHLH HES1. [score:1]
MiR-199a* (also known as miR-199a-5p, and with an identical sequence to miR-199b-5p) is also reduced in hepatocellular carcinoma [30]– [32]. [score:1]
G–I) Pre-miR-199 was cloned and transfected into Daoy cells, and three stable clones were evaluated for HES1 expression by qRT PCR and Western blotting. [score:1]
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[+] score: 17
Other miRNAs from this paper: mmu-mir-199a-1, mmu-mir-34a
Only seed sequences for miR-199a-3p and miR-34a-5p targeted the top two ranked genes Hspa1a and Dnajb1 and, their respective up-regulation and down-regulation in TOL mice were validated using quantitative RT-PCR. [score:9]
Previous studies have linked miR-199a-3p dysregulation to tumor progression in breast and liver carcinoma [44], [45] and susceptibility to hepatocyte injury [46], whereas miR-34a-5p dysregulation has been associated with the muscle condition Myotonic Dystrophy [47]. [score:3]
Figure 6 shows the IPA-generated networks associated with miRNA seed sequences miR-199a-3p and miR-34a-5p. [score:1]
Illustrated IPA-generated pathways associated with miRNA seed sequences (A) miR-199a-3p and (B) miR-34a-5p. [score:1]
In particular miRNA seed sequences miR-199a-3p and miR-34a-5p were associated with highest-ranking fold change genes Hspa1a and Dnajb1. [score:1]
0072258.g006 Figure 6Illustrated IPA-generated pathways associated with miRNA seed sequences (A) miR-199a-3p and (B) miR-34a-5p. [score:1]
Dnajb1 was associated with seed sequence miR-199a-3p (Figure 6A) whereas both Dnajb1 and Hspa1a were associated with seed sequence miR-34a-5p (Figure 6B). [score:1]
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[+] score: 17
For example, miR-146b and miR-34a were up-regulated in the liver tissues of patients with non-alcoholic steatohepatitis [11], while in the CCl [4] induced liver fibrosis, miR-199a-5p and miR-199a-3p were positively and significantly correlated to the progression of liver fibrosis [12]. [score:4]
To identify miRNAs that reflected the schistosome infections and PZQ chemotherapy, six miRNA candidates (miR-146b, miR-122, miR-223, miR-199a-5p, miR-199a-3p, miR-34a) were selected for analysis in serum that were commonly deregulated in human liver diseases. [score:4]
Expression levels of serum miR-223 (B), miR-122 (C), miR-34a (D), miR-199a-5p (E) miR-199a-3p (F), and miR-146b (G) were detected in the three groups of mice. [score:3]
The expression levels of miR-34a, miR-223, miR-122, miR-146b, miR-199a-5p, miR-199a-3p were determined using the SYBR Green Master Mix kit (TaKaRa, Dalian, China). [score:3]
Conversely, levels of serum miR-199a-3p, miR-199a-5p, and miR-146b in mice decreased after infection (Figure  1E-G). [score:1]
To test this hypothesis, we selected six candidate serum miRNAs for analysis (miR-146b, miR-122, miR-223, miR-199a-5p, miR-199a-3p, miR-34a) in the murine mo del of human schistosomiasis and then performed validation in other host species including rabbits, buffalos and human patients infected with S. japonicum. [score:1]
We analyzed the serum levels of six selected candidate miRNA molecules (miR-146b, miR-122, miR-223, miR-199a-5p, miR-199a-3p, miR-34a) from mice, rabbits, buffalos and humans infected with Schistosoma japonicum using qPCR. [score:1]
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[+] score: 16
Other miRNAs from this paper: mmu-let-7g, mmu-let-7i, mmu-mir-23b, mmu-mir-27b, mmu-mir-126a, mmu-mir-127, mmu-mir-145a, mmu-mir-181a-2, mmu-mir-182, mmu-mir-199a-1, mmu-mir-122, mmu-mir-143, mmu-mir-298, mmu-let-7d, mmu-mir-200a, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-15a, mmu-mir-23a, mmu-mir-27a, mmu-mir-31, mmu-mir-98, mmu-mir-181a-1, mmu-mir-181b-1, mmu-mir-379, mmu-mir-181b-2, mmu-mir-449a, mmu-mir-451a, mmu-mir-466a, mmu-mir-486a, mmu-mir-671, mmu-mir-669a-1, mmu-mir-669b, mmu-mir-669a-2, mmu-mir-669a-3, mmu-mir-669c, mmu-mir-491, mmu-mir-700, mmu-mir-500, mmu-mir-18b, mmu-mir-466b-1, mmu-mir-466b-2, mmu-mir-466b-3, mmu-mir-466c-1, mmu-mir-466e, mmu-mir-466f-1, mmu-mir-466f-2, mmu-mir-466f-3, mmu-mir-466g, mmu-mir-466h, mmu-mir-466d, mmu-mir-466l, mmu-mir-669k, mmu-mir-669g, mmu-mir-669d, mmu-mir-466i, mmu-mir-669j, mmu-mir-669f, mmu-mir-669i, mmu-mir-669h, mmu-mir-466f-4, mmu-mir-466k, mmu-mir-466j, mmu-mir-669e, mmu-mir-669l, mmu-mir-669m-1, mmu-mir-669m-2, mmu-mir-669o, mmu-mir-669n, mmu-mir-466m, mmu-mir-669d-2, mmu-mir-466o, mmu-mir-669a-4, mmu-mir-669a-5, mmu-mir-466c-2, mmu-mir-669a-6, mmu-mir-466b-4, mmu-mir-669a-7, mmu-mir-466b-5, mmu-mir-669p-1, mmu-mir-669a-8, mmu-mir-466b-6, mmu-mir-669a-9, mmu-mir-466b-7, mmu-mir-669p-2, mmu-mir-669a-10, mmu-mir-669a-11, mmu-mir-669a-12, mmu-mir-466p, mmu-mir-466n, mmu-mir-486b, mmu-mir-466b-8, mmu-mir-466q, mmu-mir-145b, mmu-let-7j, mmu-mir-451b, mmu-let-7k, mmu-mir-126b, mmu-mir-466c-3
For example, miR-127 has been shown to participate in cancer development [85], miR-145 has been shown to control c-Myc expression through p53 [86], miR-199a regulates MET protooncogene and affects NF-KB expression [54], miR-379 affects brain neuronal development [87], [88], miR-451 affects erythroid differentiation [89], miR-126 affects angiogenic signaling and controls blood vessel development [90], miR-143 regulates ERK5 signaling and targets KRAS gene [91], miR-298 regulates CYPA3 expression [92] and miR-486 regulates kinase activity and tumor progression [93]. [score:16]
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[+] score: 16
qRT-PCR results showed that miR-135a* was up-regulated 1.9-fold and miR-199a-5p down-regulated approximately 1.7-fold in independent experiments (Additional file 2, Figure S2). [score:7]
We chose miR-135a* and miR-199a-5p, two miRNAs that were found in microarray analyses to be highly up- and down-regulated, respectively. [score:4]
Levels of expression of the miRNAs miR-135a* and miR-199a-5p were validated by qRT-PCR. [score:3]
Click here for file Supplemental Figure S2: Validation of miR-135* and miR-199a-5a levels by qRT-PCR. [score:1]
Supplemental Figure S2: Validation of miR-135* and miR-199a-5a levels by qRT-PCR. [score:1]
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[+] score: 15
Hsa-miR-199a-5p expression is increased in human dystrophic muscle and overexpressing dre-miR-199a-5p in zebrafish muscle leads to major and lethal disruption of the myofibers [56]. [score:5]
Amongst the top-ranked miRNAs of cluster A, mmu-miR-199a– 5p (homologous to hsa-miR-199a–5p and dre-miR-199a–5p) regulates myogenic differentiation acting downstream of Srf; the latter targets multiple differentiation and proliferation factors within the Wnt signalling pathway. [score:4]
a Box-plots of miRNA expression levels of mmu-miR-18a-5p, mmu-miR-31-5p, mmu-miR-130b-3p, mmu-miR-199a-5p, mmu-miR-200c-3p, mmu-miR-224-5p in mouse quadriceps muscle at 2 days, 2 weeks, 4 weeks and 12 weeks after birth. [score:3]
A similar array has been previously conducted in a mouse mo del of muscle regeneration [73] and several miRNAs, including mmu-miR-18a-5p, mmu-miR-136-5p, mmu-miR-31-5p and mmu-miR-199a-5p, were similarly regulated as observed in our study. [score:2]
The top-ranked miRNAs for clusters A were mmu-miR-18a-5p, mmu–miR-31–5p, mmu-miR-130b–5p, mmu-miR-199a–5p, mmu-miR-200c–5p and mmu-miR-224–5p. [score:1]
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[+] score: 15
Second, it represses the expression of two COX-2 inhibiting microRNAs miR101 and miR199a and indirectly promote COX-2 expression [11]. [score:8]
We have previously demonstrated that the epithelial sodium channel (ENaC) in the endometrial epithelial cells can be activated by embryo-derived protease, which subsequently triggers a sequence of events in endometrial epithelial cells, including Ca [2+] increase, phosphorylation of CREB (Ca [2+]/cAMP responsive element binding protein), downregulation of miR101 and miR199a, upregulation of COX-2 and eventually PGE [2] production and release to the stroma, leading to decidualization and embryo implantation [10, 11]. [score:7]
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[+] score: 14
The overexpression of miR-199a in LX-2 cells triggers the upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, Col1a1, and MMP-13 mRNA [34]. [score:8]
A transcription factor, Twist-1, binds to the E-box region, regulating miR-214 and miR-199a expression [22]. [score:4]
We previously reported an increase in miR-199a in the fibrotic livers of patients with chronic HCV infection [25], and similar findings have been reported by others [31- 33]. [score:1]
miR-214 and miR-199a are encoded in a region that contains an E-box DNA promoter sequence [22]. [score:1]
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[+] score: 13
Three miRNAs (mmu-miR-378, mmu-miR-199a-3p and mmu-miR-181b) were downregulated and one (mmu-miR-23a) was upregulated in baicalin treated mice compared with UVB irradiated mice, and they were predicted to be related to DNA repair signaling pathway. [score:6]
Other potentially important miRNAs downregulated by baicalin with UV irradiation were found in this study (i. e. mmu-miR-181b, mmu-miR-199a-3p, and mmu-miR-378). [score:4]
Four miRNAs (mmu-miR-23a, mmu-miR-378*, mmu-miR-199a-3p and mmu-miR-181b) were found to be differentially expressed in the UVB group compared with the baicalin plus UVB treated group (P < 0.05). [score:2]
There are no reports associating miR-199a-3p with DNA repair and carcinogenesis. [score:1]
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[+] score: 13
To this end, inhibition of miR-100 has been shown previously to enhance perfusion recovery following FAL (Grundmann et al., 2011), and we have recently determined that miR-199a inhibition is a potent enhancer of perfusion recovery and arteriogenesis following FAL in Balb/c mice (Heuslein and Price, 2017). [score:5]
Overexpression of miR-93 (Ganta et al., 2017) or miR-199a inhibition (Heuslein et al., 2017) have both been shown to be capable of inducing arteriogenesis and improving in the ischemic downstream tissue, demonstrating such miR -based approaches are indeed possible. [score:5]
Abstract 572: inhibition of microRNA-199a-5p enhances perfusion recovery and arteriogenesis following femoral arterial ligation. [score:3]
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[+] score: 13
Three microRNAs had decreased expression in the bleomycin treated lungs (miR-26a, miR-151-3p and miR-676) while eight microRNAs had increased expression in the bleomycin treated lungs (miR-146b, miR-199a-5p, miR-21, miR-34a, miR-335-5p, miR-207, miR-301a and miR-449a). [score:5]
Further work in each of these studies demonstrated specific microRNAs (mir-21, mir-29 and mir-199a-5p) to be expressed in myofibroblasts, and to affect TGF-β signaling and fibroblast function, leading to fibrosis development. [score:4]
Finally, Lino Cardenas et al. [28] showed these four microRNAs, as well as miR-199a-5p to be among the microRNAs differentially expressed in the lungs of mice which developed fibrosis 14 days after intratracheal bleomycin instillation. [score:3]
Among those microRNAs previously shown to be perturbed in fibrosis are microRNA-21 (miR-21) [29], miR-29 [30], miR-200 [31], miR-154 [24], miR-199a-5p [28] and let-7d [26]. [score:1]
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[+] score: 13
Twist1 has been reported to regulate the expression of the miR-199a/214 cluster during development [24]. [score:5]
All results of relative expression values are shown as mean ± s. e. m. obtained from triplicate experiments (unpaired two-sample Student’s t test, * P < 0.05 and ** P < 0.01) The miR-199a/214 gene cluster is located in the Dynamin-3 (DNM3) gene as two clustered miRNAs approximately 6 kb apart. [score:3]
Lee YB Twist-1 regulates the miR-199a/214 cluster during developmentNucleic Acids Res. [score:3]
The miR-199a/214 gene cluster is located in the Dynamin-3 (DNM3) gene as two clustered miRNAs approximately 6 kb apart. [score:1]
The miR-199a/214 gene cluster is located in the 7.9-kb noncoding intron of the DNM3 gene. [score:1]
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[+] score: 13
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-16-1, hsa-mir-17, hsa-mir-19a, hsa-mir-19b-1, hsa-mir-19b-2, hsa-mir-23a, hsa-mir-26a-1, hsa-mir-26b, hsa-mir-27a, hsa-mir-29a, hsa-mir-30a, hsa-mir-31, hsa-mir-100, hsa-mir-29b-1, hsa-mir-29b-2, hsa-mir-16-2, mmu-mir-23b, mmu-mir-27b, mmu-mir-29b-1, mmu-mir-30a, mmu-mir-30b, mmu-mir-127, mmu-mir-128-1, mmu-mir-132, mmu-mir-133a-1, mmu-mir-188, mmu-mir-194-1, mmu-mir-195a, mmu-mir-199a-1, hsa-mir-199a-1, mmu-mir-200b, mmu-mir-205, mmu-mir-206, hsa-mir-30c-2, hsa-mir-30d, mmu-mir-122, mmu-mir-30e, hsa-mir-199a-2, hsa-mir-199b, hsa-mir-205, hsa-mir-211, hsa-mir-212, hsa-mir-214, hsa-mir-217, hsa-mir-200b, hsa-mir-23b, hsa-mir-27b, hsa-mir-30b, hsa-mir-122, hsa-mir-128-1, hsa-mir-132, hsa-mir-133a-1, hsa-mir-133a-2, hsa-mir-127, hsa-mir-138-1, hsa-mir-188, hsa-mir-194-1, hsa-mir-195, hsa-mir-206, mmu-mir-19b-2, mmu-mir-30c-1, mmu-mir-30c-2, mmu-mir-30d, mmu-mir-200a, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-16-1, mmu-mir-16-2, mmu-mir-23a, mmu-mir-26a-1, mmu-mir-26b, mmu-mir-29a, mmu-mir-29c, mmu-mir-27a, mmu-mir-31, mmu-mir-351, hsa-mir-200c, mmu-mir-17, mmu-mir-19a, mmu-mir-100, mmu-mir-200c, mmu-mir-212, mmu-mir-214, mmu-mir-26a-2, mmu-mir-211, mmu-mir-29b-2, mmu-mir-199b, mmu-mir-19b-1, mmu-mir-138-1, mmu-mir-128-2, hsa-mir-128-2, mmu-mir-217, hsa-mir-194-2, mmu-mir-194-2, hsa-mir-29c, hsa-mir-30c-1, hsa-mir-200a, hsa-mir-30e, hsa-mir-26a-2, hsa-mir-379, mmu-mir-379, mmu-mir-133a-2, mmu-mir-133b, hsa-mir-133b, mmu-mir-412, mmu-mir-431, hsa-mir-431, hsa-mir-451a, mmu-mir-451a, mmu-mir-467a-1, hsa-mir-412, hsa-mir-485, hsa-mir-487a, hsa-mir-491, hsa-mir-503, hsa-mir-504, mmu-mir-485, hsa-mir-487b, mmu-mir-487b, mmu-mir-503, hsa-mir-556, hsa-mir-584, mmu-mir-665, mmu-mir-669a-1, mmu-mir-674, mmu-mir-690, mmu-mir-669a-2, mmu-mir-669a-3, mmu-mir-669c, mmu-mir-696, mmu-mir-491, mmu-mir-504, hsa-mir-665, mmu-mir-467e, mmu-mir-669k, mmu-mir-669f, hsa-mir-664a, mmu-mir-1896, mmu-mir-1894, mmu-mir-1943, mmu-mir-1983, mmu-mir-1839, mmu-mir-3064, mmu-mir-3072, mmu-mir-467a-2, mmu-mir-669a-4, mmu-mir-669a-5, mmu-mir-467a-3, mmu-mir-669a-6, mmu-mir-467a-4, mmu-mir-669a-7, mmu-mir-467a-5, mmu-mir-467a-6, mmu-mir-669a-8, mmu-mir-669a-9, mmu-mir-467a-7, mmu-mir-467a-8, mmu-mir-669a-10, mmu-mir-467a-9, mmu-mir-669a-11, mmu-mir-467a-10, mmu-mir-669a-12, mmu-mir-3473a, hsa-mir-23c, hsa-mir-4436a, hsa-mir-4454, mmu-mir-3473b, hsa-mir-4681, hsa-mir-3064, hsa-mir-4436b-1, hsa-mir-4790, hsa-mir-4804, hsa-mir-548ap, mmu-mir-3473c, mmu-mir-5110, mmu-mir-3473d, mmu-mir-5128, hsa-mir-4436b-2, mmu-mir-195b, mmu-mir-133c, mmu-mir-30f, mmu-mir-3473e, hsa-mir-6825, hsa-mir-6888, mmu-mir-6967-1, mmu-mir-3473f, mmu-mir-3473g, mmu-mir-6967-2, mmu-mir-3473h
MiR-206, miR-133, miR-199, miR-100 and miR-195 were implicated in the autophagy pathway targeting BCL2, MTOR and SQSTM1 as possible autophagy gene targets (Table 6). [score:5]
However, miR-199 (p value = 0.01) and miR-200 (p value = 0.004) in contrary to the microarray results were significantly upregulated in cbs [+/–] in comparison with the control cbs [+/+] (p value < 0.05). [score:4]
MiRNAs (miR-199 and miR-200) were significantly upregulated in cbs [+/–] in comparison with the control ([*] p < 0.05, [**] p < 0.01). [score:4]
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[+] score: 13
Some miRNAs such as miR-122, miR-199 family and MiR-124 is downregulated in HCC and act as putative tumor suppressor genes [15]. [score:6]
To explore the mechanism underling the involvement of TAMs in the antitumor activity of propofol, we measured the expression of a number of miRNAs, including miR-142-3p, miR-199a, miR-122, and miR-29, which may act as tumor suppressors in HCC [23]. [score:3]
However, there was no change in the expression of miR-199a, miR-122, and miR-29 (Figure  2B). [score:3]
The levels of miR-142-3p, miR-199a, miR-122, and miR-29 were quantified with a TaqMan PCR kit. [score:1]
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In addition, miR-199a-5p is predicted to target both BChE and AChE-R, suggesting that its increase would up-regulate cholinergic stimulation in the bladder, which could also contribute to pain reactions. [score:6]
Thus miR-199a-5p is expressed in the bladder's smooth muscle and urothelium and may play a role in bladder pain syndrome (Monastyrskaya et al., 2013) by suppressing LIN7C, ARHGAP12, PALS1, RND1 and PVRL1. [score:5]
miR-199a-5p regulates urothelial permeability and may play a role in bladder pain syndrome. [score:2]
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[+] score: 13
Adenovirus -mediated p300 expression significantly increased levels of all cluster members including miR-20a (1.95-fold ±0.03, p<0.0001, Figures 4C and D), while expression of an unrelated miRNA, miR-199, was unchanged (Figure 4D), confirming that p300 gain can positively regulate expression of this anti-angiogenic microRNA cluster. [score:8]
Relative expression of miR-17∼92 cluster members and an unrelated microRNA, miR-199, in Adp300- vs AdGFP -expressing cardiac myocytes. [score:5]
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0112019.g007 Figure 7The fold upregulation of three miRNAs, miR-376a, miR-214 and miR-199a-3p, in the injury groups over the sham mice was validated using the individual real time PCR assays. [score:3]
Eight of the significantly modulated miRNAs, miR-106b, miR-199a-3p, miR-214, miR-218, miR-31, miR-434-3p, miR-671-3p, and miR-574-3p were predicted to significantly modulate several nervous system function and disease related pathways. [score:3]
The fold upregulation of three miRNAs, miR-376a, miR-214 and miR-199a-3p, in the injury groups over the sham mice was validated using the individual real time PCR assays. [score:3]
The expression profile of 3 out of 13 commonly modulated miRNAs, miR-199-3p, miR-214, and miR-376a that have been shown to be important in brain related processes and functions was also validated using the singleplex miRNA assay (Life Technologies, Carlsbad, CA) (Figure 7). [score:2]
Expression of miRNAs, miR-214, miR-376a and miR-199a-3p was validated using the singleplex miRNA assay (Life technologies, Carlsbad, CA; Assay# 002306, 001069 and 002304, respectively). [score:1]
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51
[+] score: 12
miR-143/145 along with miR-199a (another miRNA that is more highly expressed in cultured MG but absent in MG in vivo) have been recently reported to be highly expressed in cultured MG from P8 mice 25. [score:5]
Along with these studies of spinal cord injury, miR-145, together with miR-199a, has been reported to play a role in glial tumors, inhibiting migration 58 59, proliferation 60, and astrogliosis 57. [score:3]
Moreover, there were two miRNAs (miR-21 and miR-145) and three miRNAs (miR-199a-3p/5p and miR-143), which were not or barely expressed in vivo (<500 counts), but increased dramatically in vitro (over 6,900 and over 1,500 counts respectively, Fig. 5F). [score:3]
Interestingly, miR-199a is also reported to block MeCP2 function in the DGCR/Drosha complex 61. [score:1]
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[+] score: 11
Alcohol has been shown to downregulate miR-199 in rat liver sinusoidal endothelial cells (LSECs) and human endothelial cells [33]. [score:4]
MiR-27b, miR-214, miR-199a-3p, miR-182, miR-183, miR-200a, and miR-322 were found to be downregulated, whereas miR-705 and miR-1224 were increased after 4 weeks of alcohol feeding in mice [26]. [score:4]
Decreased miR-199 was associated with increased mRNA expression of endothelin-1 (ET-1) and hypoxia-inducible factor-1 α (HIF-1 α) [33]. [score:3]
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[+] score: 11
By counting eGFP-anillin expressing CMs, which could be identified according to their H2B-mCh fluorescence, increased cell cycle activity after treatment with miR-199 compared to the scramble miR control could be directly detected without further stainings (Fig.   8b). [score:3]
Further we noticed an increase in the fraction of binucleated CMs in miR-199 -treated CMs (Fig.   8g). [score:1]
Binucleation was determined as 17.7 % of total CMs for miR-199 treatment and only 9.8 % for controls. [score:1]
Ventricles from P2 mice were dissociated and the cells were transfected with either microRNA 199, (miR-199) which was reported to strongly enhance the rate of proliferation in postnatal CMs [10] or scramble miRs as a negative control. [score:1]
Surprisingly, treatment with miR-199 led to an increase of binucleated CMs, indicating induction of cell cycle variations. [score:1]
As a proof of concept, we were able to verify the proliferation-inducing effect of miR-199, as previously published [10]. [score:1]
5 µl of 500 nM Stock solutions of hsa-miR-199a-3p or miR mimic, Negative control #1 (Ambion, Life Technologies) were incubated with 0.2 µl Lipofectamine RNAi Max (Invitrogen) in 14.8 µl OPTI-MEM on 0.001 % fibronectin-coated 384-well µ-clear microtiter plates (Greiner) for 20 min at RT. [score:1]
While in transfections with scramble miRs, a basal cell cycle activity (6.6 %) could be determined, this was significantly (p = 0.0015) enhanced (19.1 %) after transfection with miR-199 (Fig.   8c). [score:1]
b Fluorescence pictures of dissociated αMHC-H2B-mCh/CAG-eGFP-anillin double transgenic hearts (P2), transfected with the cell cycle-modifying miR-199 and a miR-NC. [score:1]
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[+] score: 10
Of the remaining miRNAs, miR-199a-3p was down-regulated in (and the IL10 [−/−] mo del), but it was unaltered in the TLR5 [−/−] mo del. [score:4]
miRNA nameIL10 [−/−] mice mo del Intestinal Inflammation Inflammation mmu-miR-29b-3p x mmu-miR-122-5p x x x mmu-miR-148a-3p x mmu-miR-150-5p x x mmu-miR-192-5p x mmu-miR-194-5p x mmu-miR-146a-5p x mmu-miR-375-3p x x x mmu-miR-199a-3p x We showed that our nine-miRNA signature could discriminate between the different forms of colitis and arthritis, as well as between non-colitic mice with and without a genetic predisposition to develop the disease (WT mice versus non-colitic IL10 [−/−] mice). [score:3]
Particularly, miR-375-3p and miR-199a-3p had similar expression levels in D98 anti-TNFα treated group and non -treated group. [score:3]
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[+] score: 10
Out of these, eight miRNAs were affected after both low- and high-dose irradiation: five miRNAs (mmu-miR-33-3p, mmu-miR-200c-5p, mmu-miR-140-3p, mmu-miR-744-3p, and mmu-miR-669o-5p) were downregulated and three miRNAs (mmu-miR-152-3p, mmu-miR-199a-5p, and mmu-miR-375-3p) were upregulated. [score:7]
miR-33 inhibited high-density lipoprotein -induced radiation sensitivity in breast cancer (82), and miR-199a-5p was found to sensitize breast cancer cells to irradiation (83). [score:3]
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[+] score: 10
The miRNAs (miR-16, miR-26b, miR-101, miR-199a, miR-122 and miR-21) were selected by using miRWalk computational analyses, that covers miRNA-targets interactions information produced by 8 established miRNA prediction programs on 3' UTRs of all known genes of Human, Mouse and Rat, i. e., RNA22, miRanda, miRDB, TargetScan, RNAhybrid, PITA, PICTAR, and Diana-microT, and comparing the obtained results with data collected from the literature. [score:5]
The expression profile of six miRNAs (miR-16, miR-26b, miR-101, miR-199a, miR-122 and miR-21) was analyzed in HCC cell lines (Table 1). [score:3]
Regarding COX-2, Dey’s group [22], [23] highlighted a miRNA -mediated regulation of COX-2 by mmu-miR-101a and mmu-miR-199a* during embryo implantation and in endometrial cancer cells. [score:2]
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57
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On the basis of the opposite functions of CBX7, as oncogene and oncosuppressor, it was not surprising to find that CBX7 was able to regulate in opposite sense miRNAs that have recognized to have oncogenic functions, such as miR-199 (negatively regulated) and miR-155, miR-221 and miR-222 (positively regulated). [score:6]
The results presented in Fig.   1 confirm a drastic overexpression of miR-181, miR-137, miR-199, miR-706 and miR-719 and repression of miR-155 in Cbx7 KO MEFs in comparison with the WT ones. [score:3]
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[+] score: 9
At 4 months of high-fat diet feeding, 3 miRNAs were upregulated: let7f-5p (FC: 2.3), miR-140-5p (FC: 3.8) and miR-199-3p (FC: 2.1), but two miRNAs were down regulated in obese mice with respect to normal mice: miR-155 (FC: 1.7) and miR-322 (FC: 1.5). [score:5]
At 16 months, all 15 miRNAs were significantly downregulated in heart tissue of obese mice compared to heart tissue of normal mice: let-7f-5p (FC: 3.3), miR-10a-5p (FC: 2.6), miRNA-19b-3p (FC: 5.0), miR-25-3p (FC: 2.6), miR30e-5p (FC: 5.6), miR-140-5p (FC: 5.0), miR-155-5p (FC: 1.7), miR-146a-5p (FC: 4.0), miR-181b-5p (3.0), miR-199a-3p (FC: 3.6), miR-322 (FC: 1.5), miR-451 (FC: 1.9), miR-499-5p (FC: 5.4), miR-669m-5p (FC: 1.7) and miR-3473b (FC: 3.4). [score:3]
Based on previous pre-clinic studies, the miRNAs validated by RT-qPCR in our study are involved in alteration of glucose and lipid metabolism via insulin pathways (let-7f-5p, miR-10a-5p, miR-322) 20– 22, in cardiomyocytes apoptosis (miR-19b-3p, miR-25-3p, miR-30e-5p, miR-140-5p, miR-199a-3p, miR-499) 23– 28, in mitochondrial function (miR-181a/b) [29], in pro-inflammatory signalling (miR-146a-5p, miR-155, miR-181b-3p, miR-3473b) 30– 33, and in cardiac hypertrophy (miR-451) [34] and myocardial fibrosis process (miR-19b) 35, 36. [score:1]
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These include miR-1, miR-133, miR-30 and miR-150 which often show reduced expression, and miR-21, miR-199 and miR-214 which often show increased expression [6], [7], [8], [9], [11], [12], and they may represent miRNAs with a central role in cardiac remo delling. [score:5]
miR-199a-3p, which is co-transcribed with miR-214, was also downregulated. [score:4]
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Of the p53 -upregulated miRNAs, miR-34a/b/c-5p [39], miR-145-5p [40], miR-199a-2-3p [41], miR-34b-5p, miR-34c-5p and miR-145-5p, but not miR-34a-5p and miR-199a-2-3p, showed at least two-fold reduced expression at the Thy1- to SSEA1+ transition. [score:6]
The final submodule, 2C, is found only in the FDR5 dataset and is composed of only four miRNAs (miR-199a-5p, miR-145-5p, miR-155-5p, miR-143-5p), including two differentiation -associated miRNAs (miR-145-5p, miR-155-5p), and tends to have significant decreases in expression early in reprogramming. [score:3]
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[+] score: 9
rno-miR-novel-8, rno-homolog-miR-26, and rno-homolog-miR-199 miRNAs were selected from Tier 1, and rno-miR-sno-57 miRNA was selected from Tier 2. In addition, we analysed the expression of miR-741-3p and miR-743a-3p and found that, in accordance with sequencing data, they were highly expressed in rat PSCs. [score:5]
rno-homolog-miR-26 and rno-homolog-miR-199 miRNAs were expressed in EFs, ESCs and iPSCs, which is consistent with the data obtained from sequencing. [score:3]
Four novel miRNAs with the following coordinates: chrX:−:151288045–151288101 (rno-miR-novel-8); chr7:+:70463555–70463594 (rno-homolog-miR-26); chr3:+:16697111–16697143 (rno-homolog-miR-199); and chr18:−:69422790–69422857 (rno-miR-sno-57) were selected for the validation. [score:1]
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Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-16-1, hsa-mir-17, hsa-mir-21, hsa-mir-22, hsa-mir-28, hsa-mir-29b-1, hsa-mir-16-2, mmu-let-7g, mmu-let-7i, mmu-mir-1a-1, mmu-mir-29b-1, mmu-mir-124-3, mmu-mir-9-2, mmu-mir-133a-1, mmu-mir-145a, mmu-mir-150, mmu-mir-10b, mmu-mir-195a, mmu-mir-199a-1, hsa-mir-199a-1, mmu-mir-200b, mmu-mir-206, mmu-mir-143, hsa-mir-10a, hsa-mir-10b, hsa-mir-199a-2, hsa-mir-217, hsa-mir-218-1, hsa-mir-223, hsa-mir-200b, mmu-let-7d, hsa-let-7g, hsa-let-7i, hsa-mir-1-2, hsa-mir-124-1, hsa-mir-124-2, hsa-mir-124-3, hsa-mir-133a-1, hsa-mir-133a-2, hsa-mir-143, hsa-mir-145, hsa-mir-9-1, hsa-mir-9-2, hsa-mir-9-3, hsa-mir-150, hsa-mir-195, hsa-mir-206, mmu-mir-200a, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-16-1, mmu-mir-16-2, mmu-mir-21a, mmu-mir-22, mmu-mir-29c, rno-let-7d, rno-mir-329, mmu-mir-329, rno-mir-331, mmu-mir-331, rno-mir-148b, mmu-mir-148b, rno-mir-135b, mmu-mir-135b, hsa-mir-200c, hsa-mir-1-1, mmu-mir-1a-2, mmu-mir-10a, mmu-mir-17, mmu-mir-28a, mmu-mir-200c, mmu-mir-218-1, mmu-mir-223, mmu-mir-124-1, mmu-mir-124-2, mmu-mir-9-1, mmu-mir-9-3, mmu-mir-7b, mmu-mir-217, hsa-mir-29c, hsa-mir-200a, hsa-mir-365a, mmu-mir-365-1, hsa-mir-365b, hsa-mir-135b, hsa-mir-148b, hsa-mir-331, mmu-mir-133a-2, mmu-mir-133b, hsa-mir-133b, rno-let-7a-1, rno-let-7a-2, rno-let-7b, rno-let-7c-1, rno-let-7c-2, rno-let-7e, rno-let-7f-1, rno-let-7f-2, rno-let-7i, rno-mir-7b, rno-mir-9a-1, rno-mir-9a-3, rno-mir-9a-2, rno-mir-10a, rno-mir-10b, rno-mir-16, rno-mir-17-1, rno-mir-21, rno-mir-22, rno-mir-28, rno-mir-29b-1, rno-mir-29c-1, rno-mir-124-3, rno-mir-124-1, rno-mir-124-2, rno-mir-133a, rno-mir-143, rno-mir-145, rno-mir-150, rno-mir-195, rno-mir-199a, rno-mir-200c, rno-mir-200a, rno-mir-200b, rno-mir-206, rno-mir-217, rno-mir-223, dre-mir-7b, dre-mir-10a, dre-mir-10b-1, dre-mir-217, dre-mir-223, hsa-mir-429, mmu-mir-429, rno-mir-429, mmu-mir-365-2, rno-mir-365, dre-mir-429a, hsa-mir-329-1, hsa-mir-329-2, hsa-mir-451a, mmu-mir-451a, rno-mir-451, dre-mir-451, dre-let-7a-1, dre-let-7a-2, dre-let-7a-3, dre-let-7a-4, dre-let-7a-5, dre-let-7a-6, dre-let-7b, dre-let-7c-1, dre-let-7c-2, dre-let-7d-1, dre-let-7d-2, dre-let-7e, dre-let-7f, dre-let-7g-1, dre-let-7g-2, dre-let-7h, dre-let-7i, dre-mir-1-2, dre-mir-1-1, dre-mir-9-1, dre-mir-9-2, dre-mir-9-4, dre-mir-9-3, dre-mir-9-5, dre-mir-9-6, dre-mir-9-7, dre-mir-10b-2, dre-mir-16a, dre-mir-16b, dre-mir-16c, dre-mir-17a-1, dre-mir-17a-2, dre-mir-21-1, dre-mir-21-2, dre-mir-22a, dre-mir-22b, dre-mir-29b-1, dre-mir-124-1, dre-mir-124-2, dre-mir-124-3, dre-mir-124-4, dre-mir-124-5, dre-mir-124-6, dre-mir-133a-2, dre-mir-133a-1, dre-mir-133b, dre-mir-133c, dre-mir-143, dre-mir-145, dre-mir-150, dre-mir-200a, dre-mir-200b, dre-mir-200c, dre-mir-206-1, dre-mir-206-2, dre-mir-365-1, dre-mir-365-2, dre-mir-365-3, dre-let-7j, dre-mir-135b, rno-mir-1, rno-mir-133b, rno-mir-17-2, mmu-mir-1b, dre-mir-429b, rno-mir-9b-3, rno-mir-9b-1, rno-mir-9b-2, rno-mir-133c, mmu-mir-28c, mmu-mir-28b, hsa-mir-451b, mmu-mir-195b, mmu-mir-133c, mmu-mir-145b, mmu-mir-21b, mmu-let-7j, mmu-mir-21c, mmu-mir-451b, mmu-let-7k, rno-let-7g, rno-mir-29c-2, mmu-mir-9b-2, mmu-mir-124b, mmu-mir-9b-1, mmu-mir-9b-3
For example, miR-199a was specifically expressed in neural tissues. [score:3]
Cortex let-7c-1, miR-10a, miR-21, miR-124a-1, miR-128a, miR-135b, miR-150, miR-199a, miR-217, miR-329, miR-451. [score:1]
Hypothalamus miR-17, miR-29c, miR-124a-1, miR-128a, miR-150, miR-199a, miR-217, miR-223, miR-329, miR-429. [score:1]
Olfactory bulb let-7b, let-7c-1, let-7c-2, miR-10a, miR-16, miR-17, miR-21, miR-22, miR-28, miR-29c, miR-124a-1, miR-124a-3, miR-128a, miR-135b, miR-143, miR-148b, miR-150, miR-199a, miR-206, miR-217, miR-223, miR-29b-1, miR-329, miR-331, miR-429, miR-451. [score:1]
Dorsal root ganglion let-7c, miR-17, miR-145, miR-150, miR-199a, miR-223, miR-365, miR-451. [score:1]
Brain stem let-7c-1, miR-17, miR-135b, miR-150, miR-199a, miR-218-1, miR-223, miR-329. [score:1]
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63
[+] score: 8
Here, we intended to identify suitable MREs for bladder cancer specific adenovirus -mediated TRAIL expression from the miRNAs with downregulated expression in bladder cancer, including miR-1 [18- 21], miR-99a [22], miR-100 [23], miR-101 [24, 25], miR-125b [23, 26, 27], miR-133a [18, 20, 21, 23, 28- 30], miR-143 [22, 23, 31- 33], miR-145 [21, 23, 29- 31, 34], miR-195-5p [35], miR-199a-3p [36], miR-200 [37, 38], miR-203 [39, 40], miR-205 [37], miR-218 [21, 41], miR-490-5p [42], miR-493 [43], miR-517a [44], miR-574-3p [45], miR-1826 [46] and let-7c [42]. [score:8]
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[+] score: 8
More recently, miR-199a-3p was also shown to be downregulated and target mTOR in hepatocarcinoma cells [30]. [score:6]
These characteristics of miR-100 and miR-199a-3p are quite similar to those of miR-99a, indicating that mTOR expression might be regulated redundantly by various closely related miRNAs. [score:2]
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65
[+] score: 8
For example, hsa-miR-133a, hsa-miR-200b, hsa-miR-206, and hsa-miR-218 were considered as tooth tissue-specific miRNAs [4]; eight differentially expressed miRNAs were expressed during morphogenesis and seven were expressed in the incisor cervical loop containing the stem cell niche [1]; the three most highly expressed microRNAs in dental epithelium were identified as mmu-miR-24, mmu-miR-200c, and mmu-miR-205, while mmu-miR-199a-3p and mmu-miR-705 were found in dental mesenchyme [2]; and miR-200 was suggested to play an important role in the formation of incisor cervical loop during stem cell–fueled incisor growth [5]. [score:8]
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66
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A closer look at these interactions showed that, for instance, miR-199a targets the homologs of esrrga and rb1. [score:3]
As in the case of miR-199a, miR-195 also targets esrrg. [score:3]
We also identified 18 (unique) computationally-inferred interactions between our hubs and other heart regeneration miRs: hsa-miR-1, hsa-miR-195 and hsa-miR-199a 51. [score:1]
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67
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Further, lncRNA-Map2k4 may regulate neuronal proliferation and apoptosis through a miR-199a/FGF1 pathway (Lv, 2017), and lncRNA Meg3 acts as a functional regulator in the regulation of the PTEN/PI3K/AKT signaling cascade, during the process of synaptic plasticity in neurons (Tan et al., 2017). [score:4]
lncRNA-Map2k4 sequesters miR-199a to promote FGF1 expression and spinal cord neuron growth. [score:3]
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We validated by RT-qPCR analysis the down-regulation of Let-7i-5p and miR-199a-3p and the up-regulation of miR-4284 during the conversion of white to brite adipocytes (Fig. 1C). [score:7]
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69
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All of them were downregulated: mmu-miR-1 and -805 at ST, mmu-miR-199a-3p, -200a and -429 at IT and mmu-miR-27a and -200b at LT, except mmu-miR-206 which was upregulated at LT. [score:7]
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70
[+] score: 7
Similarly, when pri-mir-21, pri-mir-199a, and pri-mir-381 primary transcripts were up-regulated, their mature miRNA forms were up-regulated as well (Figure 6). [score:7]
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71
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Other miRNAs from this paper: mmu-mir-146a, mmu-mir-199a-1, mmu-mir-301a, mmu-mir-34a
Although in our results we demonstrated that overexpression of miR-301a-3p repressed SMAD4 expression through direct binding to its 3′UTR, there were still other miRNA consensus in the SMAD4 3′UTR (e. g., sites for miR-34a, miR-146a, and miR-199a, which have been identified as negative regulators of SMAD4 in gastric cancer and glioblastoma) [40, 41, 42]. [score:7]
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72
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Among the up regulated miRNAs in STHdh [Q111]/Hdh [Q111] cells, expressions of miR-214, miR-299 and miR-335 were also up regulated in three of the four cell mo dels and expression of miR-199a was increased in two cell mo dels. [score:7]
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73
[+] score: 7
Similarly, we observed that miR-199a is up-regulated in liver fibrosis [39] but down-regulated in HCC [40]. [score:7]
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74
[+] score: 7
Other miRNAs from this paper: mmu-mir-181a-2, mmu-mir-199a-1, mmu-mir-181a-1
In hepatocellular carcinoma, SNHG12 promoted tumorigenesis and metastasis by acting as an endogenous miR-199a/b-5p sponge to regulate the expression of MLK3 and affect the NF-κB pathway [27]. [score:4]
Similarly, a previous study also revealed SNHG12 de-suppress mixed-lineage protein kinase 3 (MLK3) by sponging miR-199a/b-5p in hepatocellular carcinoma [27]. [score:3]
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75
[+] score: 7
Other miRNAs from this paper: mmu-mir-199a-1
How the expression of CAV1 is regulated remains unclear; Li et al. demonstrated that CAV1 is a direct target of miR199a-5p in hepatocytes. [score:7]
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76
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Of these, eight (miR-193b, miR-199a-3p/hsa-miR-199b-3p, miR-455-3p, miR-210, miR-381 (also known as miR-381-3p), miR-92a, miR-320c, and miR-136) were upregulated, while the other four (miR-490-5p, miR-4287, miR-BART8*, and miR-US25-1*) were downregulated during this process [22]. [score:7]
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77
[+] score: 6
Using the Patrocles database, we found polymorphic miRNA target sites for bta-miR-199b, -miR-199a-5p, and -miR-361 in the IL1B gene and for –miR-126 in the CYP11B1 gene. [score:3]
Interestingly, the expression of -miR-199b, -miR-199a-5p and –miR-126 in the bovine mammary gland has already been experimentally confirmed. [score:3]
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78
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In particular miR-23b, miR-199a, and miR-15a displayed increased expression during early AVC development and characterization of target genes suggests that they are involved in regulating epithelial-mesenchymal transition (EMT) signaling pathways [106]. [score:5]
Bonet F. Duenas A. Lopez-Sanchez C. Garcia-Martinez V. Aranega A. E. Franco D. Mir-23b and mir-199a impair epithelial-to-mesenchymal transition during atrioventricular endocardial cushion formation Dev. [score:1]
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79
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Axl expression is also negatively regulated by the microRNAs (miR) miR-34 and miR-199a/b, with an inverse correlation between AXL protein levels and miR-34a expression found in a panel of cancer cell lines [53]. [score:6]
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80
[+] score: 6
Nine miRNAs (miR-148a-3p, miR-183a-5p, miR-214-3p, miR-27a-3p, miR-92a-3p, miR-378a-3p, miR-23a-3p, miR-21a-5p and miR-16-5p) were upregulated, and four (miR-155-5p, miR-199a-3p, miR-320-3p and miR-125a-5p) were downregulated in exosomes from RANKL -induced RAW 264.7 cells compared with RAW 264.7 cells (Figure 1f and Supplementary Figure S1d). [score:6]
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81
[+] score: 6
Other miRNAs from this paper: mmu-mir-199a-1, mmu-mir-148a, mmu-mir-503
Interestingly, IKKβ is the target of miR148a, miR503, and miR199a, 45, 46, 47 which are upregulated microRNAs in vascular calcification. [score:6]
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82
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3) SM genes are indirectly and negatively controlled by SRF through SM miRNAs; we previously found that a large number of SM miRNAs, such as miR-143/miR-145 and miR-199a/miR-214, are SRF targets [26], and SM miRNAs target SM genes (see Table S5). [score:6]
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83
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Hydrogen regulates expressions of miR-9, miR-21, and miR-199, and modifies expressions of IKK-β, NF-κB, and PDCD4 in LPS-activated retinal microglia cells [64]. [score:6]
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84
[+] score: 6
Comparison of the miRNA expression profiles between the control and SFN treated samples revealed a number of miRNA including miR-214, miR-145, miR-199a, and miR-199b that were significantly upregulated in SFN -treated H460 cells and were reported to be involved in tumorigenesis and progression (Supplementary Figure 3A & 3B). [score:6]
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85
[+] score: 6
No differences in miRNA expression were found in kidneys of ischemic heart failure mice compared to control animals (S6 Table) and only small differences were observed between expression levels of miR-18a-5p, miR-30e-5p, miR-199a-3p and miR-223-3p in the LV of mice with ischemic heart failure compared to controls (S7 Table), however not reaching significance after Bonferroni correction for multiple testing. [score:3]
In addition to the cardiac specific miR-208a-3p and miR-499-5p, we found that the expression of let-7i-5p, miR-16-5p, miR-27a-3p, miR-199a-3p and miR-223-3p was significantly higher in the heart compared to the kidney, independent of the presence of ischemic heart failure (S4 Fig and S5 Table). [score:2]
In general, the rank order of the expression levels of the measured miRNAs was comparable in mice and rats, with the highest miRNA levels of miR-16-5p and miR-223-3p and the lowest levels of miR-199a-3p, miR-652-3p, miR-423-3p and miR-26b-5p (S1– S3 Figs). [score:1]
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86
[+] score: 6
Other miRNAs from this paper: mmu-let-7g, mmu-let-7i, mmu-mir-124-3, mmu-mir-140, mmu-mir-141, mmu-mir-152, mmu-mir-182, mmu-mir-183, mmu-mir-191, mmu-mir-199a-1, mmu-mir-200b, mmu-mir-205, mmu-let-7d, mmu-mir-200a, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-96, mmu-mir-200c, mmu-mir-214, mmu-mir-199b, mmu-mir-124-1, mmu-mir-124-2, mmu-mir-7a-1, mmu-mir-7a-2, mmu-mir-7b, dre-mir-7b, dre-mir-7a-1, dre-mir-7a-2, dre-mir-182, dre-mir-183, dre-mir-199-1, dre-mir-199-2, dre-mir-199-3, dre-mir-205, dre-mir-214, dre-mir-430a-1, dre-mir-430b-1, dre-mir-430c-1, mmu-mir-429, mmu-mir-449a, dre-mir-429a, dre-let-7a-1, dre-let-7a-2, dre-let-7a-3, dre-let-7a-4, dre-let-7a-5, dre-let-7a-6, dre-let-7b, dre-let-7c-1, dre-let-7c-2, dre-let-7d-1, dre-let-7d-2, dre-let-7e, dre-let-7f, dre-let-7g-1, dre-let-7g-2, dre-let-7h, dre-let-7i, dre-mir-7a-3, dre-mir-96, dre-mir-124-1, dre-mir-124-2, dre-mir-124-3, dre-mir-124-4, dre-mir-124-5, dre-mir-124-6, dre-mir-140, dre-mir-141, dre-mir-152, dre-mir-200a, dre-mir-200b, dre-mir-200c, dre-mir-430c-2, dre-mir-430c-3, dre-mir-430c-4, dre-mir-430c-5, dre-mir-430c-6, dre-mir-430c-7, dre-mir-430c-8, dre-mir-430c-9, dre-mir-430c-10, dre-mir-430c-11, dre-mir-430c-12, dre-mir-430c-13, dre-mir-430c-14, dre-mir-430c-15, dre-mir-430c-16, dre-mir-430c-17, dre-mir-430c-18, dre-mir-430a-2, dre-mir-430a-3, dre-mir-430a-4, dre-mir-430a-5, dre-mir-430a-6, dre-mir-430a-7, dre-mir-430a-8, dre-mir-430a-9, dre-mir-430a-10, dre-mir-430a-11, dre-mir-430a-12, dre-mir-430a-13, dre-mir-430a-14, dre-mir-430a-15, dre-mir-430a-16, dre-mir-430a-17, dre-mir-430a-18, dre-mir-430i-1, dre-mir-430i-2, dre-mir-430i-3, dre-mir-430b-2, dre-mir-430b-3, dre-mir-430b-4, dre-mir-430b-6, dre-mir-430b-7, dre-mir-430b-8, dre-mir-430b-9, dre-mir-430b-10, dre-mir-430b-11, dre-mir-430b-12, dre-mir-430b-13, dre-mir-430b-14, dre-mir-430b-15, dre-mir-430b-16, dre-mir-430b-17, dre-mir-430b-18, dre-mir-430b-5, dre-mir-430b-19, dre-mir-430b-20, dre-let-7j, mmu-mir-449c, mmu-mir-449b, dre-mir-429b, mmu-let-7j, mmu-let-7k, mmu-mir-124b
By contrast, we identified 12 miRNAs corresponding to 9 families (miR-199, miR-140, miR-152, miR-214, miR-205, miR-200, miR-183, miR-182, miR-96) that displayed highly enriched expression in the olfactory system (Figure 1A). [score:3]
Five of 24 miRNAs, including miR-199a [∗] and miR-140 [∗] (Figure 2A, center column, and Table S3), showed expression in the mesenchyme underlying or cartilage surrounding the MOE and VNO. [score:3]
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87
[+] score: 6
Other miRNAs from this paper: mmu-mir-199a-1, mmu-mir-18a, mmu-mir-199b, mmu-mir-18b
Recent association studies between miRNA markers and lung cancer development have demonstrated that the miRNAs miR-18, miR-199, and miR-519c can suppress HIF-1α expression for the purpose of assessing cancer prognosis [39– 41]. [score:6]
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88
[+] score: 6
Recently, we reported that the expression of hES cell-specific miRNAs miR-302 d, miR-372 and miR-367 and miR-200c, as well as miR-199a, were strongly up-regulated by activin A [6]. [score:6]
[1 to 20 of 1 sentences]
89
[+] score: 5
In man, high expression of miR-423, miR-199a-3p, miR-93*, and miR-377 in plasma[17] and low expression of miR-146a-5p and miR-155in whole blood can predict acute GvHD[18]. [score:5]
[1 to 20 of 1 sentences]
90
[+] score: 5
miR-199a functions as an onco-suppressor targeting the oncogene Met, therefore impairing Met -mediated invasive growth of cells [43]. [score:5]
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91
[+] score: 5
Elevated expression of miR-182 [39], miR-429 [40], miR-199a, 199a*, 200a, and 200b was positively and significantly correlated to the progression of liver fibrosis [41]. [score:3]
Our analysis also suggests that 6 DEmiRs, miR-17-5p, miR-486b-5p, miR-19a-3p, miR-484, miR-199a-5p and miR-182-5p, are likely to be co-regulated by both Ctcf and Spi1. [score:2]
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92
[+] score: 5
Other miRNAs from this paper: mmu-mir-199a-1, mmu-mir-31, mmu-mir-330, mmu-mir-378b
Overexpression of miR-199a-5p inhibits keratinocyte migration [10]. [score:5]
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93
[+] score: 5
In contrast, increased miR-199a-5p was found in NASH by inhibiting nuclear receptor corepressor 1 translation[31]. [score:5]
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94
[+] score: 5
NFATs may also control genes encoding signaling molecules as variate as Ca [2+] regulators [inositol 1,4,5-trisphosphate (IP [3]) receptor (IP [3]R), regulator of calcineurin 1 (RCAN1)], growth factors (VEGF, neurotrophins), myelination genes (P0 and Krox-20), glucose regulation genes (insulin, HNF1, PDX, and GLUT2), cell cycle and death regulator/activators [p21 [Waf1], c-Myc, cyclin -dependent kinase 4 (CDK4), B-cell lymphoma 2 (Bcl-2), and cyclins A2, D1, and D2], oncogenes (Wnt, β-catenin), microRNAs (miR-21, miR-23, miR-24, miR-27, miR-125, miR-195, miR-199, and miR-224), and surfactants (sftpa, sftpb, sftpc, and abca3) [9, 65– 74]. [score:5]
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95
[+] score: 5
Only 15 miRNAs from the 64 DEMs had validated target genes in IPA by target filter analysis, including miR-6349, miR-101a-3p, miR-6394, miR-126a-3p, miR-721, miR-143-3p, miR-497a-5p, miR-93-5p, miR-215-5p, miR-199a-3p, miR-23a-3p, miR-27b-3p, miR-2861, miR-30a-5p, and miR-370-3p. [score:5]
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96
[+] score: 5
The top ten highly expressed miRNAs (ranked by the mean expression value of three tissues) are miR-199a-3p, miR-199a-5p, miR-146a-5p, miR-146b-5p, miR-125a-5p, miR-200a-3p, miR-200b-3p, miR-142a-5p, miR-486b-5p and miR-182-5p. [score:5]
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97
[+] score: 5
Chakrabarty et al. found that mmu-miR-101a and mmu-miR-199a* were spatiotemporally expressed in the mouse uterus during implantation concurrently with the expression of the cyclooxygenase-2 gene, which is critical for embryo implantation [4]. [score:5]
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98
[+] score: 5
In this study we have focused on miR-148a, however L5-miRNA targeting could be applied to other miRNAs, such as miR-122, let-7 or miR-199, which have also shown great success in excluding gene expression from the hepatocytes in E1A-miRNA adenoviruses [5, 7, 19- 21]. [score:5]
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99
[+] score: 5
Other miRNAs from this paper: hsa-let-7a-1, hsa-let-7a-2, hsa-let-7a-3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1, hsa-let-7f-2, hsa-mir-15a, hsa-mir-17, hsa-mir-18a, hsa-mir-19a, hsa-mir-19b-1, hsa-mir-20a, hsa-mir-24-1, hsa-mir-24-2, hsa-mir-27a, hsa-mir-92a-1, hsa-mir-92a-2, mmu-let-7g, mmu-let-7i, mmu-mir-1a-1, mmu-mir-15b, mmu-mir-23b, mmu-mir-27b, mmu-mir-130a, mmu-mir-133a-1, mmu-mir-140, mmu-mir-24-1, hsa-mir-196a-1, mmu-mir-199a-1, hsa-mir-199a-1, mmu-mir-200b, mmu-mir-206, hsa-mir-30c-2, hsa-mir-196a-2, hsa-mir-199a-2, hsa-mir-199b, hsa-mir-200b, mmu-mir-301a, mmu-let-7d, hsa-let-7g, hsa-let-7i, hsa-mir-1-2, hsa-mir-15b, hsa-mir-23b, hsa-mir-27b, hsa-mir-130a, hsa-mir-133a-1, hsa-mir-133a-2, hsa-mir-140, hsa-mir-206, mmu-mir-30c-1, mmu-mir-30c-2, mmu-mir-196a-1, mmu-mir-196a-2, mmu-mir-200a, mmu-let-7a-1, mmu-let-7a-2, mmu-let-7b, mmu-let-7c-1, mmu-let-7c-2, mmu-let-7e, mmu-let-7f-1, mmu-let-7f-2, mmu-mir-15a, mmu-mir-18a, mmu-mir-20a, mmu-mir-24-2, mmu-mir-27a, mmu-mir-92a-2, hsa-mir-200c, hsa-mir-1-1, mmu-mir-1a-2, mmu-mir-17, mmu-mir-19a, mmu-mir-200c, mmu-mir-199b, mmu-mir-19b-1, mmu-mir-92a-1, hsa-mir-30c-1, hsa-mir-200a, hsa-mir-301a, mmu-mir-133a-2, mmu-mir-133b, hsa-mir-133b, hsa-mir-196b, mmu-mir-196b, dre-mir-196a-1, dre-mir-199-1, dre-mir-199-2, dre-mir-199-3, hsa-mir-18b, dre-let-7a-1, dre-let-7a-2, dre-let-7a-3, dre-let-7a-4, dre-let-7a-5, dre-let-7a-6, dre-let-7b, dre-let-7c-1, dre-let-7c-2, dre-let-7d-1, dre-let-7d-2, dre-let-7e, dre-let-7f, dre-let-7g-1, dre-let-7g-2, dre-let-7h, dre-let-7i, dre-mir-1-2, dre-mir-1-1, dre-mir-15a-1, dre-mir-15a-2, dre-mir-15b, dre-mir-17a-1, dre-mir-17a-2, dre-mir-18a, dre-mir-18b, dre-mir-18c, dre-mir-19a, dre-mir-20a, dre-mir-23b, dre-mir-24-4, dre-mir-24-2, dre-mir-24-3, dre-mir-24-1, dre-mir-27a, dre-mir-27b, dre-mir-27c, dre-mir-27d, dre-mir-27e, dre-mir-30c, dre-mir-92a-1, dre-mir-92a-2, dre-mir-92b, dre-mir-130a, dre-mir-133a-2, dre-mir-133a-1, dre-mir-133b, dre-mir-133c, dre-mir-140, dre-mir-196a-2, dre-mir-196b, dre-mir-200a, dre-mir-200b, dre-mir-200c, dre-mir-206-1, dre-mir-206-2, dre-mir-301a, dre-let-7j, hsa-mir-92b, mmu-mir-666, mmu-mir-18b, mmu-mir-92b, mmu-mir-1b, dre-mir-196c, dre-mir-196d, mmu-mir-3074-1, mmu-mir-3074-2, hsa-mir-3074, mmu-mir-133c, mmu-let-7j, mmu-let-7k, dre-mir-24b
miR-199a, a bone morphogenic protein 2-responsive MicroRNA, regulates chondrogenesis via direct targeting to Smad1. [score:5]
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100
[+] score: 4
MiR-101a and miR-199a* regulate uterine expression of cyclooxygenase-2 around the implantation site [15]. [score:4]
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