100 publications mentioning mmu-mir-195a

Open access articles that are associated with the species Mus musculus and mention the gene name mir-195a. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

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Supported by miRNA target analysis and luciferase reporter assays, we propose that miR-195-5p targets the 3′-UTR of YAP1 mRNA and inhibits YAP expression, leading to proliferative inhibition in CRC cells.

Moreover, upregulation of miR-195-5p suppresses proliferation, migration, invasion, and EMT of colon cancer cells through targeting the YAP1 mRNA 3′-UTR.

Our results demonstrated that miR-195-5p was a potent suppressor of YAP1, and miR-195-5p -mediated downregulation of YAP1 significantly reduced tumor development in a mouse CRC xenograft mo del.

Thirdly, miR-195-5p can directly regulate expression of YAP1 by targeting its 3′-UTR.

The presumed targets of integrated-signature miRNAs, especially the most significant has-miR-195, were identified by four different target prediction algorithms: TargetScan v7.1 (http://www.

Downregulation of miR-195-5p is concomitant with upregulation of YAP1 in primary human CRC.

Additionally, consistent with the proposed regulations of miR-195-5p on the Hippo pathway and EMT, expression patterns of YAP, Ki67, Vimentin, ZEB2, and E-cadherin in miR-195-5p-agomir -treated tumors were similar to the in vitro results, substantiating the tumor suppressor function of miR-195-5p in CRC tumorigenesis (Fig.   7e–g).

YAP1 expression was downregulated after miR-195-5p treatment by qRT-PCR analysis and western blot.

Additionally, expressions of TAZ, Vimentin, ZEB2, and SMAD3 protein were also negatively regulated by miR-195-5p, while E-cadherin was positively regulated by miR-195-5p, suggesting the negative regulations of miR-195-5p on the Hippo pathway and EMT in CRC cells.

Hence, major players in this pathway may be tightly regulated by miR-195-5p in the colorectum, and miR-195-5p restoration could target YAP/TAZ/EMT pathway to suppress CRC progression.

The multicenter sequencing of 1893 carcinoma/normal paired samples revealed that miR-195-5p was a tumor-suppressing miRNA and was downregulated in tumor tissues [10].

Furthermore, the present study also shed light on the potential role of miR-195-5p in CRC metastasis, where ectopic expression of miR-195-5p could reduce cell migration and invasion and promote expression of the EMT marker, E-cadherin.

Cell culture and transfection of miR-195 mimic, inhibitor, and siRNA of target gene.

We transfected DLD1 and HCT116 cells with miR-195-5p mimic or miR mimic NC, and miR-195-5p inhibitor or miR-195-5p inhibitor NC, separately.

The results showed that miR-195-5p suppressed luciferase activity whereas miR-195-5p inhibitor could promote luciferase activity for the reporter plasmid carrying wild-type YAP1 3′-UTR (Fig.   6c, P < 0.05), but no significant effects were observed for the reporter plasmid carrying mutant YAP1 3′-UTR (i. e., pmiR-RB-REPORT [TM]-mut-YAP1-3′-UTR).

Fig. 4Ectopic expression of miR-195-5p inhibits proliferation and colony formation of DLD1 and HCT116 cells.

In contrast, miR-195-5p inhibitor treatment could lift this inhibition (Fig.   4a–b).

As miR-195 showed a consistent pattern of downexpression in human CRC, we wondered about the expression pattern of miR-195-5p in the colon cancer cell lines.

Since predicted targets of miR-195-5p were enriched for the Hippo/YAP signaling pathway, we hypothesize that miR-195-5p may function by targeting the YAP1 mRNA.

The miR-195-5p -expressing cells migrated toward the wound at a much slower rate than the control or the cells treated by miR-195 inhibitors (Fig.   5a–b).

Cells were washed by phosphate-buffered saline (PBS, pH 7.4) before transient transfection with 50 nM miR-195-5p mimic or miR mimic NC, 100 nM miR-195-5p inhibitor or miR inhibitor NC.

Bold italics indicate statistically significant values (P < 0.05) Significantly, in spite of the presence of biological and technical confounding factors, such as the differences in sample cohorts, treatments, and microarray technologies, in all eight independent CRC expression microarrays, our results indicated the importance of miR-195 expression in human CRC (Fig.   2d).

f Kaplan–Meier analysis of OS based on YAP1 mRNA levels in 60 cases of CRC patients Since the Hippo pathway emerged in our previous KEGG analysis for the miR-195 targets, it is conceivable that miR-195 may directly regulate this pathway during CRC tumorigenesis.

e Venn diagrams of putative miR-195 targets predicted by TargetScan (v7.1), miRDB, PicTar, and DIANA-TarBase (v7.0).

Fig. 7Ectopic expression of miR-195-5p suppresses tumor growth in vivo.

These results suggest that miR-195-5p binds directly to the predicted binding site(s) in the YAP1 3′-UTR and negatively regulates YAP1 expression.

f Kaplan–Meier analysis of OS based on YAP1 mRNA levels in 60 cases of CRC patients Since the Hippo pathway emerged in our previous KEGG analysis for the miR-195 targets, it is conceivable that miR-195 may directly regulate this pathway during CRC tumorigenesis.

Representative photomicrographs (a) and quantifications (b) of BrdU staining in DLD1 and HCT116 cells after transfection with miR-195-5p mimic, miR-195-5p mimic NC, miR-195-5p inhibitor, or miR-195-5p inhibitor NC for 48 h. Bar = 100 μm.

We next examined the pathophysiological significance of miR-195-5p downregulation in CRC and its underlying regulatory mechanisms.

Hsa-miR-195 mimic and mimic negative control (NC), hsa-miR-195 inhibitor, and inhibitor negative control (NC) were purchased from RiboBio Co.

Both qRT-PCR and western blotting revealed that the YAP1 level was reduced in miR-195-5p -expressing cells, while its level was restored in miR-195-5p inhibitor -treated cells (Fig.   6d–f).

Four downregulated miRNAs (hsa-let-7a, hsa-miR-125b, hsa-miR-145, and hsa-miR-195) can be potentially useful diagnostic markers in the clinic.

Only hsa-let-7a, hsa-miR-125b, hsa-miR-145, and hsa-miR-195 were significantly downregulated in CRC tumors (Fig.   1c), and each of these miRNAs could provide a high accuracy on CRC tissue classification as estimated by ROC curve analysis (Fig.   2a; Additional file 10: Figure S1a).

We showed for the first time that the full-length 3′-UTR of the human YAP1 mRNA was a direct target of miR-195-5p.

In addition, we validated and obtained 4 downregulated miRNAs (i. e., hsa-let-7a, hsa-miR-125b, hsa-miR-145, and hsa-miR-195) from over 250 tumors from the TCGA-COAD database which granted us the opportunity to systematically analyze the potential molecular mechanisms associated with the pathophysiology of CRC.

d Forest plots summarizing the downregulation of hsa-miR-195 from eight datasets in the integrated analysis.

Thus, given the results of the integrated analysis and the in vitro experiments, we hypothesize that the decreased expression of miR-195-5p may promote CRC progression and development.

The downregulation of miR-195 has been consistently observed in the CRC patients with poor overall survivals, indicating that miR-195 may be functionally important in CRC pathogenesis.

Four downregulated miRNAs (hsa-let-7a, hsa-miR-125b, hsa-miR-145, and hsa-miR-195) were demonstrated to be potentially useful diagnostic markers in the clinical setting.

We investigated the mechanism of miR-195-5p -mediated regulations on colon cancer cells and found that over -expression of miR-195-5p markedly inhibited cellular proliferation and invasion of CRC cells.

The luciferase reporter plasmid pmiR-RB-REPORT [TM]-YAP1-3′-UTR or mutant reporter plasmid carrying point mutations in the putative miR-195-5p binding sites was co -transfected with miR-195-5p mimics or miR mimic NC and inhibitors, separately.

Thus, negative values indicate downregulation of miR-195 in CRC.

In the transwell invasion and migration assay, we found that invasion and migration of the miR-195-5p -expressing cells was reduced, and this effect could be reversed by the miR-195-5p inhibitor (Fig.   5c–f).

In our study, we confirmed that miR-195 was downregulated in CRC tissues, which was significantly correlated with poor survivals of CRC patients.

c Kaplan–Meier curves of the overall survivals (OS) reveal that downregulated miR-195-5p is associated with poor prognosis in CRC patients.

Taken together, these results indicate that deregulated miR-195 expression plays a critical role in human CRC.

MiR-195-5p targets human YAP1 and inhibits YAP1 in colon cancer cells.

Representative photomicrographs (c) and quantifications (d) of the colony formation assay after transfection with miR-195-5p mimic, miR-195-5p mimic NC, miR-195-5p inhibitor, or miR-195-5p inhibitor NC for 8 days.

We also identified the Hippo signaling pathway as being enriched in the prediction analysis of the miR-195-5p targets.

In previous analysis, the Hippo signaling pathway was significantly enriched for the predicted targets of miR-195-5p (P = 6.47E-05) (Additional file 13: Figure S2).

Meanwhile, the protein-protein interaction network (PPI) suggested that YAP1 is the hub gene of the miR-195 targets (Additional file 14: Figure S3).

c Kaplan–Meier plots for overall survival for a discriminatory median miR-195 expression, from TCGA sequencing data to assess prognostic accuracy.

Functional enrichment and target prediction of miR-195.

YAP1, which harbors two conserved miR-195-5p cognate sites, namely, 162-168 and 1857–1862 of YAP1 3′-UTR (Fig.   6b), is a predicted target of miR-195-5p.

HCT116 treated with miR-195-5p inhibitor showed mesenchymal features.

The most significantly enriched by the predicted targets of miR-195 (P = 6.47E-05).

The expression of miR-195 was validated in The Cancer Genome Atlas (TCGA) datasets, and an independent validation sample cohort.

Additionally, Kaplan–Meier and Cox’s proportional hazards regression mo del survival analysis revealed that patients with low expression levels of miR-195-5p had shorter overall survival (Fig.   3c, Table  2).

To our knowledge, this is the first meta-analysis that reveals the detailed mechanism where loss of miR-195-5p leads to malignant progression of CRC via unleashed expression of YAP1.

Furthermore, it is likely that other molecules or signaling pathways will be discovered that are also targeted by miR-195-5p in CRC.

Protein-protein interaction network of the consensus target gene of miR-195-5p.

Overexpression of miR-195-5p in DLD1 and HCT116 cells repressed cell growth, colony formation, invasion, and migration.

Aberrant expression of hsa-miRNA-195 (miR-195) which is distinguished as a clinically noteworthy miRNA has previously been observed in multiple cancers, yet its role in CRC remains unclear.

Interestingly, the top KEGG pathways enriched for the miR-195 targets were mainly associated with cancer-specific pathways (Additional file 12: Table S9), including the Hippo signaling pathway, proteoglycans in cancer, viral carcinogenesis, pathways in cancer, and prostate cancer (Fig.   2f).

Firstly, reduced expression of miR-195-5p in primary CRC was revealed independently by both integrated analysis and TCGA-COAD validation analysis, suggesting miR-195-5p can be an effective diagnostic and prognostic marker in the clinical setting.

Ectopic expression of miR-195-5p could significantly reduce proliferation, migration, invasion, and EMT in two colon cancer cell lines, DLD1 and HCT116.

Our study may serve as rational for targeting the miR-195-5p/YAP interaction in a novel therapeutic application to medicate CRC patients.

Interestingly, we also found that loss of miR-195-5p accelerated YAP expression and promoted nuclear accumulation of YAP and EMT in vitro [19, 55, 56].

Therefore, miR-195-5p significantly inhibits the tumorigenicity of DLD1 cells in vivo.

We have demonstrated that miR-195-5p is dramatically downregulated in human CRC tissues compared with normal colorectal tissues.

Red frame shows the predicted miR-195 targets.

To confirm the tumor suppressor role of miR-195-5p in vivo, we established a BALB/c nude mouse xenograft mo del using DLD1 cells.

Secondly, ectopic expression of miR-195-5p in colon cancer cell lines considerably decreased cell growth, migration, and invasion.

a Expression of miR-195-5p in two colon cancer cell lines, DLD1 and HCT116, and the normal NCM460 cells.

HCT116 cell lines were showed in cell morphology after transfection miR-195-5p inhibitor (10 nM) after 7–10 days.

We identified miR-195-5p binding sites within the 3’-untranslated region (3′-UTR) of the human yes -associated protein (YAP) mRNA.

These results indicated that the anti-cancer and reverse EMT efficacy of miR-195-5p is partly attributed to its inhibitory role on YAP, which was confirmed by qRT-PCR and western blot of YAP1 (Additional file 16: Figure S5f-g) in DLD1 cells.

Inhibition of miR-195-5p function contributed to aberrant cell proliferation, migration, invasion, and epithelial mesenchymal transition (EMT).

Collectively, miR-195-5p suppresses tumor cell growth in vitro and tumorigenicity in vivo.

a An ROC curve built on a univariate classification mo del based on miR-195 expression across independent TCGA dataset for predicting CRC.

Fig. 5Ectopic expression of miR-195-5p in DLD1 and HCT116 cells reduces cell migration and invasion.

c Relative luciferase activity of reporter plasmids carrying wild-type or mutant YAP1 3′-UTR in DLD1 and HCT116 cells co -transfected with negative control (NC) or miR-195-5p mimic or miR-195-5p inhibitor.

Enrichment analysis of predicted miR-195 targets in KEGG cell signaling pathway database.

The results showed that miR-195-5p expression in the tumors was significantly (P < 0.05) reduced in the tumors relative to PANT (Fig.   3b).

We conducted target prediction for validated miR-195 with high-stringency.

Fig. 3Expressions of miR-195-5p and YAP1 are inversely correlated in primary CRC tumors.

For this purpose, we searched different databases for the potential targets of miR-195-5p that exhibited oncogenic properties.

Finally, miR-195-5p suppressed tumor growth in vivo.

These data offer a plausible mechanism accounting for the tumor-suppressing function of miR-195-5p, further supporting the notion that the miR-195-5p/YAP1/EMT axis can be the therapeutic focus for eliminating CRC tumorigenesis [19].

Soon et al. found aberrant expression of miR-195 could indicate a poorer prognosis in adrenocortical carcinoma [39].

Colon cancer cells were transfected with miR-195 mimic and inhibitor, after which cell proliferation, colony formation, migration, invasion, and dual luciferase reporter were assayed.

Only the low level of miR-195 in tumors was substantially correlated with reduced overall survivals of CRC patients, suggesting miR-195 may negatively regulate CRC progression.

MiR-195 as a tumor suppressor has been reported in various types of cancer.

f Top ten KEGG pathways that are enriched for the miR-195 targets are mainly cancer-specific pathways To further investigate whether the deregulated miRNAs correlate with the survivals of the CRC patients, we performed Kaplan-Meier and Cox’s proportional hazards regression mo del analysis and found that low miR-195 level was significantly correlated with poor overall survivals of CRC patients (Fig.   2c, Table  2), suggesting the prognostic value of miR-195-5p in clinical CRC diagnosis.

f Top ten KEGG pathways that are enriched for the miR-195 targets are mainly cancer-specific pathways To further investigate whether the deregulated miRNAs correlate with the survivals of the CRC patients, we performed Kaplan-Meier and Cox’s proportional hazards regression mo del analysis and found that low miR-195 level was significantly correlated with poor overall survivals of CRC patients (Fig.   2c, Table  2), suggesting the prognostic value of miR-195-5p in clinical CRC diagnosis.

MiR-195-5p inhibits proliferation and colony formation of colon cancer cells.

BrdU incorporation assay revealed that miR-195-5p inhibited DNA synthesis in DLD1 and HCT116 cells (Fig.   4a–b).

Significantly, the miR-195-5p level was inversely correlated with the YAP1 level in the tumors as calculated by Pearson’s correlation (R [2] = 0.531, P = 4.02E − 11) (Fig.   3e), suggesting that miR-195-5p may downregulate YAP1 in CRC (Fig.   3e).

MiR-195-5p inhibits migration and invasion of colon cancer cells.

So far, the results suggest that miR-195-5p negatively regulates YAP1 levels in CRC cells.

MiR-195-5p suppresses tumor growth in vivo.

These results, taken together, clearly demonstrate that miR-195-5p negatively regulates invasion and migration of colon cancer cells.

In addition, clonogenic assay showed that miR-195-5p mimic treatment decreased the clonogenic survivals of DLD1 and HCT116 cells compared with blank controls, while miR-195-5p inhibitor -treated DLD1 and HCT116 cells showed a reversed phenotype (Fig.   4c–d), suggesting miR-195-5p negatively regulates cancer cell proliferation.

Similarly, we found a lower expression of miR-195-5p in DLD1 and HCT116 cell lines, compared with the normal intestinal epithelium cell line NCM460 (Fig.   3a).

CRC patients with a decreased level of miR-195-5p in tumor tissues had significantly shortened survival as revealed by the TCGA colon adenocarcinoma (COAD) dataset and our CRC cohort.

We confirmed that the miR-195-5p level was high in patients with favorable survival in TCGA, and may play a critical role in CRC tumorigenesis.

The diamonds represent overall, combined mean difference for miR-195.

To assess the prospective functions of the most significant has-miR-195, we discharged the Kyoto Encyclopedia of Genes and Genomes (KEGG) using the Database for Annotation, Visualization and Integrated Discovery (DAVID) [25].

b The 3′-UTR of YAP1 mRNA harbors two miR-195-5p cognate sites.

Xenograft mouse mo dels were used to determine the role of miR-195 in CRC tumorigenicity in vivo.

Hsa-miRNA-195(miR-195) YAP1 Colorectal cancer (CRC) Epithelial mesenchymal transition (EMT) Prognosis Biomarker Colorectal cancer (CRC) has been the fourth leading cause of cancer death worldwide for several decades [1].

We first examined the effect of miR-195-5p on proliferation of DLD1 and HCT116 cells.

e Scatter plots showing the inverse association between miR-195-5p and YAP1 mRNA levels.

MiR-195-5p agomir or miR agomir NC (RiboBio Co.

We distinguished miR-195-5p as a clinically noteworthy miRNA in CRC as found in lung cancer [18].

In the clinic, miR-195-5p can serve as a prognostic marker to predict the outcome of the CRC patients.

Both the volumes and weights of the tumors treated with miR-195-5p agomir were significantly reduced relative to those treated with miR agomir NC (Fig.   7a–d).

Future work will focus on revealing additional functions of miR-195-5p in CRC carcinogenesis and progression.

For the qRT-PCR detection of mature miR-195-5p expression, we purchased the Bulge-Loop™ miRNA qRT-PCR Primer Set and Control Primer Set (RiboBio, Guangzhou, China).

<5 μg/mL) 0.982(0.542–1.780) 0.953  Hsa-miR-195 (≥median vs.

Luo et al. found miR-195 might tender a novel tactic for the diagnosis and treatment of breast cancer patients [41].

<5 μg/mL) 0.893(0.506-1.573) 0.695  Hsa-miR-195 (≥median vs.

Our integrated microRNA profiling approach identified miR-195-5p independently associated with prognosis in CRC.

Li et al. found that extracellular vesicles delivering miR-195 to intrahepatic cholangiocarcinoma could decrease the tumor size and improve survivals in a rat mo del [40].

Starting day 8 post-implantation, we injected miR-195-5p agomir or miR agomir NC intratumorally every 4 days for 7 treatments.

The conversion from epithelial cells to mesenchymal cells, characterized by spindle-type cell morphology, was observed in HCT116 cells treated with miR-195-5p inhibitor (Additional file 15: Figure S4).

We assessed the role of miR-195-5p on the migration and invasion of DLD1 and HCT116 cells.

Among them, only miR-195 is inversely correlated with overall survival in CRC patients.

Experimentally, we verified that the Hippo pathway core effector YAP1 mRNA 3′-UTR had two conserved miR-195-5p cognate sites.

In addition, we performed KEGG pathway analysis to elucidate the potential biological functions of miR-195 integrated-signature.

Supporting this notion, a colorimetric based cellular proliferation assay (i. e., CCK8 assay) showed consistent phenotypes when treating with miR-195-5p mimic or its inhibitor (Fig.   4e–f).

g Immunohistochemistry of tumor tissues treated with or without miR-195-5p.

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372 inhibition -dependent reduction in ACC and FAS expression and impaired intracellular triglyceride contents could be largely restored by miR-195 suppression (Fig.   5c, Supplementary Fig.   6b, Supplementary Fig.   7a, b), suggesting that reduced miR-195 suppression of ACC and FAS expression plays a key role in uc.

372 specifically suppresses miR-195/miR-4668 expression by binding to pri-miR-195/pri-miR-4668 to relieve miR-195/miR-4668 -mediated suppression of functional target genes such as ACC, FAS, SCD1, and CD36, leading to lipid accumulation in hepatocytes.

372 drives hepatic steatosis through inhibition of miR-195/miR-4668 maturation to relieve miR-195/miR-4668 -mediated suppression of functional target gene related to lipid synthesis, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1), and genes related to lipid uptake such as CD36, leading to hepatic lipid accumulation.

372 inhibits the maturation of miR-195/miR-4668 to regulate expression of ACC, FAS, SCD1, and CD36.

b Expression levels of ACC and FAS in the HepG2 cells transfected with miR-195 mimic and inhibitor for 24 h in the presence with Ad-uc.

372 was overexpressed, no changes in pri-miR-195/pri-miR-4668, pre-miR-195/pre-miR-4668, and mature miR-195/miR-4668 expression were identified (Supplementary Fig.   5b).

a Expression levels of ACC and FAS in the HepG2 cells transfected with miR-195 mimic and inhibitor at a final concentration of 20 nM for 48 h (n = 3).

372 drives hepatic steatosis by repressing the maturation of miR-195/miR-4668, thus relieving the suppression of target genes, including ACC, FAS, SCD1, and CD36.

Additionally, miR-195 suppresses cell proliferation, invasion, and metastasis in breast cancer cells by targeting FAS, HMGCR, ACC, and CYP27B1 [26].

Based on the previous studies and our observations, we consider that FAS and ACC/SCD1 and CD36 are direct targets of miR-195/miR-4668 mediated by Ago2.

Singh R Yadav V Kumar S Saini N Microrna-195 inhibits proliferation, invasion and metastasis in breast cancer cells by targeting fasn, hmgcr, acaca and cyp27b1Sci.

Intriguingly, we observed an increase of pri-miR-195/pri-miR-4668 and a reduction of miR-195/miR-4668 expression in the liver of NAFLD patients (Fig.   8f, g).

372 affected lipid metabolism through miR-195/miR-4668 -mediated target genes.

Previous studies have shown that ACC and FAS are target genes of miR-195 [26] (Supplementary Fig.   6a).

372 inhibits the maturation of miR-195/miR-4668.

Abnormal expression of miR-195 has been wi dely reported in diverse biological systems 51, 52.

For instance, miR-195 can bind the 3′UTRs of cyclin D1, CDK6, and E2F3, thereby suppressing cell proliferation in human hepatocellular carcinoma cells [52].

372, we determined expression levels of pri-miR-195/pri-miR-4668, pre-miR-195/pre-miR-4668, and miR-195/miR-4668 upon uc.

372i-infected and miR-195 inhibitor -transfected HepG2 cells in the presence with with 300 μM O/P mixture for 48 h (representative blots from three similar experiments) (n = 3).

As shown by real-time PCR analysis, miR-195 markedly suppressed mRNA levels of FAS and ACC, even when uc.

Consistently, we found that miR-195 markedly suppresses mRNA levels of FAS and ACC, even when uc.

In the present study, the sequence alignment results in Supplementary Fig.   6a, b showed canonical complementarity between miR-195/miR-4668 and their respective targets, which is necessary for the formation of the miRNA–mRNA duplex [56].

372 on abnormal lipid accumulation is subjected to regulation of miR-195/ miR-4668 in the liver of NAFLD patients.

372 was functionally involved in the abnormal hepatic lipid accumulation of NAFLD patients by regulation of pri-miR-195/pri-miR-4668 processing.

Liu L Chen L Xu Y Li R Du X Microrna-195 promotes apoptosis and suppresses tumorigenicity of human colorectal cancer cellsBiochem.

372 regulates pri-to-pre-miRNA cleavage of miR-195 and 4668 by binding pri-miR-195/pri-miR-4668.

372 is subject to regulation by miR-195/miR-4668 in the steatotic liver of NAFLD patients.

372 binds to pri-miR-195/pri-miR-4668.

372 and the terminal loop region of pri-miR-195/pri-miR-4668.

How miR-195/miR-4668 link to the metabolic genes such as FAS, ACC, SCD1, and CD36?

f The levels of pri-miR-195, pre-miR-195, and mature miR-195 in the liver samples of NAFLD patients or healthy control (n = 11).

283 + A impairs microprocessor recognition and efficient pri-miR-195 cropping [22].

TRIzol reagent (Invitrogen) was used to isolate the precipitated RNA and the level of pri-miR-195 or pri-miR-4668 was further analyzed using real-time PCR.

372. e The levels of pri-miR-195/pri-miR-4668, pre-miR-195/pre-miR-4668, and mature miR-195/miR-4668 in the HepG2 cells transfected with Ad-uc.

Subsequently, we analyzed the hepatic levels of pri-miR-195/pri-miR-4668 and mature miR-195/miR-4668 in these patients.

372 significantly increased the level of pri-miR-195/pri-miR-4668 in the immunoprecipitated RNA with antibody against Drosha, indicating a binding between uc.

Of note, pri-miR-195 and pri-miR-4668 displayed a complementarity with the ultraconserved region of uc.

To further assess that pri-miR-195/pri-miR-4668 interacts with uc.

This complementarity involved nucleotides located at the terminal loop region site within the miR-195 and miR-4668 primary transcripts (Fig.   4d).

To explore the interaction between miR-195/miR-4668 and uc.

372 could bind pri-miR-195/pri-miR-4668 in HepG2 cells transfected with Ad-uc.

372 at the terminal loop region site within the miR-195 and miR-4668 primary transcript.

Based on the reduced levels of miR-195 in HepG2 cells treated with an O/P mixture (Supplementary Fig.   6a), we investigated the effects of miR-195 on expression of ACC and FAS.

d The stem-loop sequence of pri-miR-195 (left panel) and pri-miR-4668 (right panel), and their partial complementarity with uc.

372 in the HepG2 increased the levels of pri-miR-195/pri-miR-4668 and repressed the maturation of miR-195/miR-4668 (Fig.   4e).

Of note, significantly reduced pri-miR-195/pri-miR-4668 and elevated pre-miR-195/pre-miR-4668 and mature miR-195/miR-4668 were observed in the HepG2 cells transfected with Ad-372i (Supplementary Fig.   5a).

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Relatively robust expression of miR-195 and miR-221 was detected in the cultured cells, while miR-497 expression was significantly lower.

The authors acknowledge the limitation of a small number of mice with nodal metastases (n = 3), however despite this the specificity of miR-10b’s role in tumour invasion in this study was further validated by the fact that this up-regulation did not hold true for miR-195,497 and 221.

In contrast circulating miR-195 has been reported to be significantly upregulated in patients with breast cancer [23].

MiR-195 and miR-497 expression was observed to increase again at week 6, however this was not significant.

Similar finding have also been reported in a recent study examining miR-195 and miR-497 in both primary human breast tumours and various breast cancer cell lines, highlighting their inhibitory role in breast cancer [22].

The expression of a panel of breast cancer associated miRNAs (miR-10b, miR-221, miR-195 and miR-497) was examined on the basis of their reported relevance [29], [30].

Furthermore there was no significant relationship between the expression of circulating miR-221, miR-195 and miR-497 at week 6 and final tumour volume detected.

Breast cancer specific blood -based miRNAs such as miR-195 have been shown to be upregulated in cancer patients compared to controls and return to normal levels post tumour excision [14].

RNA was extracted from cultured MDA-MB-231 cells, reverse transcribed and RQ-PCR carried out targeting miR-195, miR-497, miR-221, and miR-10b.

MiR-221, miR-195, and miR-497 were detected in all samples and expression was found to remain unchanged in healthy controls throughout the 6 week duration of the study.

Summary statistics and graphical techniques were used to summarise and compare the change in mean log expression level for each response variable (i. e. miR-497, miR-195 and miR-221) between the groups (i. e. Control, MFP and SC) and across time (i. e. Weeks 1, 3 and 6).

The mean log miR-195 expression at Week 1 is higher compared to Week 3 (p<0.001) and to Week 6 (p = 0.002) with no evidence of a significant difference between Weeks 3 and 6 (p = 0.98).

Although no direct relationship between circulating miRNAs levels and tumour burden was observed, a significant positive correlation was observed between miR-497 and miR-195 within tissue.

MiR-195 and miR-497 were both detected in circulation of all animals however were not significantly altered in expression in tumour bearing animals compared to controls at termination of the study.

At 6 weeks following tumour induction there was no significant difference in miR-221 (Figure 3A), miR-195 (Figure 3B), or miR-497 (Figure 3C) expression at a circulating level in tumour bearing animals (n = 15) compared to healthy controls (n = 5).

The expression levels of miR-195 and miR-497 in MFP and SC cancer tissues were significantly decreased when compared to healthy tissue (p<0.05, p<0.001, Figure 2D, E) respectively.

0050459.g005 Figure 5Further significant positive correlations were observed between circulating miR-195 and miR-497, circulating miR-195 and miR-221, and between miR-497 and miR-221.

Circulating miR-195 and miR-497 was significantly decreased at week 3 following tumour induction (B, C).

Circulating levels of miR-195, miR- 497 and miR-221 were analysed and correlated with tissue levels from the same animals.

Prior to in vivo inoculation, expression of miR-10, miR-221, miR-195 and miR-497 was investigated in the cultured MDA-MB-231 cell line.

Across all blood samples at each time point (total n = 60), a significant positive correlation was detected between miR-195 & miR-497 in the circulation (r = 0.56, p<0.001, Figure 5).

MiR-195 and miR-497 have been shown to both originate from the miR-16 super family [54].

Further significant positive correlations were observed between circulating miR-195 and miR-497, circulating miR-195 and miR-221, and between miR-497 and miR-221.

MiR-195 and 497 have been shown to be significantly down regulated in breast tumour tissue [22].

When analysing miRNA release over the 6 week study it was observed that both miR-195 and miR-497 were significantly decreased at week 3 following tumour induction.

There was a significant positive correlation between miR-497 and miR-195 detected in all tissues examined of the murine mo del (r = 0.61, p<0.001, Figure 2F).

Further significant correlations were detected between miR-221 and miR-497 (r = 0.4, p<0.001, Figure 5) and miR-221 and miR-195 in the circulation (r = 0.4, p<0.05, Figure 5).

There was evidence of a possible interaction between group and time where mean log miR-195 expression for the SC group compared to the controls at week 3 compared to week 1 (p = 0.02, Figure 4).

4

The exosomes showed marked expression of 6 (miR-16, miR-17, miR-20a, miR-20b, miR-26a, and miR-26b) of the 10 miRNA targets at 8 h after CLP; the expression of the remaining 4 miRNA targets (miR-106a, miR-106b, miR-195, and miR-451) increased; however, the increase was not significant (Fig. 5B).

The miRNA targets that were significantly up-regulated in the CLP experiment, as shown by the microarray experiments, are shown in Table 1. The expressions of 2 (miR-16 and miR-17), 6 (miR-20a, miR-16, miR-17, miR-451, miR-106a, and miR-106b), and 7 miRNAs (miR-26b, miR-20b, miR-17, miR-20a, miR-106a, miR-26a, and miR-195) increased significantly in the whole blood of mice at 4, 8, and 24 h after CLP, respectively.

miR-195 may also exert its tumor suppressive effects by decreasing the expression of multiple NF-κB downstream effectors via direct targeting of IKKα and TAB3 [48].

Two miRNAs (miR-20b and miR-195) were only significantly expressed in the CLP mo del, but were not expressed after injection of E. coli.

In this study, we demonstrated that experimental sepsis induced by CLP caused time -dependent upregulation of the circulating miRNAs miR-16, miR-17, miR-20a, miR-20b, miR-26a, miR-26b, miR-106a, miR-106b, miR-195, and miR-451.

miR-195 has a complementary site in the 3′-UTR of a membrane water channel protein Aquaporin 8 (AQP8), thereby modulating AQP8 expression [46].

In this study, 2 miRNAs (miR-20b and miR-195) were significantly expressed in the CLP mo del, but not after exposure to E. coli.

Among these miRNAs, the levels of miR-195 and miR-451 detected in the Ago2 immunoprecipitates were more than 20-fold that in the controls and the levels of miR-16, miR-20a, miR-26a, and miR-106b were more than 10-fold that in the control.

5

Finally, the fact that consistent expression of five genes in circulating miRNA expression profiles exists from multiple mouse strains, at different ages, and with different disease mo dels provides strong evidence that miR-146a, miR-16, miR-195, miR-30e, and miR-744 are useful as circulating miRNA endogenous references.

Furthermore, miR-146a and miR-16 are highly expressed in serum, while miR-30e, miR-195, and miR-744 have relatively lower expression.

As shown in Figure 2, miR-146a and miR-16 are highly expressed in serum, while miR-30e, miR-195, and miR-744 have lower expression levels.

As shown in Figure 2, these five serum miRNAs, miR-146a, miR-16, miR-195, miR-30e, and miR-744 were stably expressed in mouse regardless of strain, age, and disease condition.

We preformed normalization of single qRT-PCR targets from the related mouse disease mo del using miR-146a, miR-16, and miR-195.

We have found five miRNAs, miR-146a, miR-16, miR-195, miR-30e, and miR-744 to be stably expressed in all tested strains across different ages and conditions.

Cross normalization of these references to each other revealed that miR-16 and miR-195 are extremely stable endogenous references in this particular mouse mo del (our unpublished data).

These 72 miRNAs were candidate endogenous controls, following a series of statistical analyses only five genes (miR-146a, miR-16, miR-195, miR-30e, and miR-744) passed the criteria of ANOVA p>0.3, SD<1, and pair-wise |ΔΔ C [T]|<0.5.

0031278.g002 Figure 2−ΔC [T] values of miR-146a, miR-16, miR-195, miR-30e, and miR-744 show stability across all samples (w = weeks).

Notably, these five miRNAs, miR-16, miR-744, miR-195, miR-146a, and miR-30e share a 100% identity between human and mouse [32], [33], [34], [35].

−ΔC [T] values of miR-146a, miR-16, miR-195, miR-30e, and miR-744 show stability across all samples (w = weeks).

6

significantly decreased when the expression of down-regulated miR-195 was recovered (pooled MD = [-3.10]; 95% confidence interval [CI]: [-5.60]- [-0.59]; p = 0.04; Figure 9, part 1) [62, 64].

significantly decreased when the expression of down-regulated miR-195 was recovered (pooled MD = [-2.21]; 95% confidence interval [CI]: [-3.19]- [-1.24]; p = 0.35;) [62, 64]; there were 28 mice in the intervention arm and 28 in the control arm.

As we could see in Figure 9, the effects on inhibiting tumor volume were most significant when the aberrantly expressed miR-34a, miR-143 and miR-214 were corrected, and then followed by miR-195 and miR-133a Figure 9 SD, standard deviation; CI, confidence interval.

As we could see in Figure 4A, the effect on inhibiting tumor weight was most significant when the aberrantly expressed oncogene miR-214 was corrected, and followed by rescuing miR-143, then miR-195 or miR-34a.

As we could see in Figure 9, the effects on inhibiting tumor volume were most significant when the aberrantly expressed miR-34a, miR-143 and miR-214 were corrected, and then followed by miR-195 and miR-133a Figure 9 SD, standard deviation; CI, confidence interval.

This resulted 5 different miRNAs were analyzed, including 4 tumor suppressor miRNAs(miR-195, miR-143, miR-34a and miR-133) and 1oncogene(miR-214).

This resulted 4 different miRNAs were anylyzed, including 3 tumor suppressor miRNAs(miR-195, miR-143 and miR-34a) and 1 oncogene(miR-214).

The efficacy due to up -regulating miR-195 or miR-34a was comparable.

7

Taken together with our later observations that targeting of the liver-specific miR-122-5p or poorly abundant miR-195-5p, miR-25-3p, miR-200a/b/c-3p, miR182-5p and the mutant miR-224-5p mut2 by 2′OMe AMOs (but not their LNA/DNA AMO counterparts) also resulted in significant inhibition of immunostimulatory ssRNA sensing, our work establishes sequence -dependent and miRNA-independent off-target inhibitory activity of 2′OMe AMOs on the immune sensing of pathogenic RNA by human and mouse phagocytes.

The sequence-specific and miRNA-independent significant inhibition of immunostimulatory ssRNA sensing by 2′OMe AMOs targeting miR-195-5p, miR-25-3p, miR-122-5p, miR-200a/b/c-3p and miR182-5p (Figure 2B) was supported by the lack of inhibitory activity with LNA/DNA AMOs (Figure 2C), and the low abundance of these miRNAs (less than 100-fold the level of the most abundant miRNA in BMMs) (Figure 2A).

The inhibitory effect was dose dependent, with a maximum IC [50] around 2 nM for miR-195-5p 2′OMe AMO (Supplementary Figure S1), while other 2′OMe AMOs (for example, miR-224-5p, not shown) showed only minor inhibitory activity when used at concentrations as high as 80 nM.

Next, dose-response studies comparing the activity of highly inhibitory AMOs (miR-182-5p 2′OMe and miR-331-3p LNA/DNA) and that of poorly inhibitory AMOs (miR-224-5p 2′OMe and miR-195-5p LNA/DNA) on both TLR7 and TLR8 sensing were conducted in human PBMCs (Figure 3C and D).

Critically, this core sequence overlapped with a significantly enriched motif found in all the inhibitory sequences of Class 2 AMOs previously identified, in 5′-3′ orientation (for miR-200a/b-3p, and miR-25-3p) or 3′-5′ orientation (for AMO-NC1, miR-182-5p, miR-122-5p and miR-195-5p) (Figure 4C and Supplementary Table S2).

In line with a predominant TLR7 inhibitory effect of LNA/DNA AMOs, the miR-331-5p LNA/DNA AMO significantly decreased IFN-α levels compared to the miR-195-5p LNA/DNA AMO, while having a smaller impact on TLR8 -driven TNF-α production (Figure 3D).

Ordinary one-way (A and B) or two-way (C and D) ANOVA with Dunnett's multiple comparison tests to the RD+B-406AS-1 (A and B), the miR-224-5p+B-406AS-1 (C) or the miR-195-5p+B-406AS-1 (D) conditions are shown.

Similar results were seen with miR-195-5p 2′OMe AMO dose-response studies (Supplementary Figure S1).

8

At 40 weeks of age, the expression of miR-216, miR-217, miR-223, miR-141, miR-483-3p (p-value = 0.031), miR-195, Let-7b (p-value = 0.063) and miR-96 were significantly downregulated; on the other hand, the expression of miR-21, miR-205, miR-146b (p-value = 0.031), and miR-34c (p-value = 0.063) were upregulated in KC mice compared to the control animals (Figure 2C).

At 30 weeks of age, the expression of miR-216 (p-value = 0.016), miR-217 (p-value = 0.0078), miR-150 (p-value =0.023), Let-7b (p-value = 0.031,) and miR-96 were significantly downregulated, whereas the expression of miR-146b (p-value = 0.0078), miR-205, (p-value - 0.0078), miR-21, miR-195 (p-value = 0.031), and miR-34c (p-value = 0.063) were significantly upregulated in KC animals compared to control animals (Figure 2B).

On the other hand, miR-146b, miR-34c, miR-223, miR-195 (p-value = 0.031) and miR-216 (p-value = 0.063) were downregulated in KC mice compared to control littermates.

9

Additionally, cardiac overexpression of miR-195 results in pathological cardiac growth and heart failure in transgenic mice [53], so that modulation of miR-15 miRNAs during development and maturation are unlikely to allow specific conclusions about the effect of miR-15 miRNAs on elastin metabolism.

We then proceeded to test the effect of treating RFL-6 cells with miR-29 or miR-195 mimics on the expression of Eln, Col1a1, and Col1a2.

In conclusion, we have shown that miR-29 and the miR-15 family members miR-195 and miR-497 are differentially regulated between the newborn and the six-week old murine aorta, and using in vitro assays we have demonstrated that they regulate elastin by means of multiple MREs in both the 3′ UTR and the CDS.

Computational analysis revealed eleven additional 7–8mer binding sites for miR-29 in the coding sequence (CDS) of elastin (Fig. 4A, dashed lines), as well as eight MREs with perfect complementarity to the seed sequence of the miR-15 family members miR-195/miR-497 (Fig. 4A, arrows).

The first such construct contains three miR-497/miR-195 MREs but no miR-29 MREs, and showed repression by the miR-497 precursor but not by the miR-29 precursor.

Therefore, some genes have a lower number of miR-497 MREs in comparison to the number of miR-195 MREs.

revealed eleven additional 7–8mer binding sites for miR-29 in the coding sequence (CDS) of elastin (Fig. 4A, dashed lines), as well as eight MREs with perfect complementarity to the seed sequence of the miR-15 family members miR-195/miR-497 (Fig. 4A, arrows).

miR-29 and the miR-15 family members miR-195/miR-497 form three intergenic clusters in mouse chromosomes 1, 6, and 11..

The finding of a total of 14 MREs for miR-29 as well as 13 for the miR-15 miRNA miR-195 in the coding and 3′ UTR sequence of Eln is highly statistically significant (Fig. 6), and to our knowledge a similar finding has not been previously reported for any miRNA.

miR-195 and miR-497 share the same seed sequence at nucleotides 2–8 but differ in position one.

3′ UTR and CDS MREs for miR-29, miR-195, and miR-497 in the elastin gene.

Although a secondary effect of miR-195 cannot be excluded, this result is consistent with biologically active MREs for miR-195 in the CDS of the elastin gene.

Mimics for miR-29 or miR-195 led to repression of elastin expression as measured by qPCR (Fig. 5), and treatment of the cells with antagonists of miR-29 had the opposite effect (Supplementary Fig. S3).

As shown in Fig. 3 of the main manuscript, the first nucleotide of the seed sequences of miR-195 and miR-497 is different ().

Triplicate qPCR analyses for Col1a1, Col1a2, and Eln following treatment of RFL-6 cells with scrambled (black), miR-29 (dark grey), or miR-195 (light grey) precursor.

0016250.g005 Figure 5Triplicate qPCR analyses for Col1a1, Col1a2, and Eln following treatment of RFL-6 cells with scrambled (black), miR-29 (dark grey), or miR-195 (light grey) precursor.

10

Almeida et al. demonstrated that miR-195 in human primary mesenchymal stromal/stem cells negatively regulated proliferation, osteogenesis and had paracrine effect on angiogenesis by targeting vascular endothelial growth factor (VEGF) 25.

Our results indicate that aptamer -mediated activation of miR-195 expression in endothelial cells promoted type H vessels formation and osteogenesis in aged mice, suggesting a novel strategy to treat age-related bone loss and senile osteoporosis.

We used qRT-PCR to detect the expression of miR-497 and miR-195.

Among them, the expression of miR-497 and miR-195, which are found clustered at the same locus, is 3 times higher in CD31 [hi]Emcn [hi] than in CD31 [lo]Emcn [lo] endothelial cells (Fig. 1a).

MiR-497 and miR-195 belong to miR-15 family, thus we also tested the expression of other members of the miR-15 family in bone marrow endothelial cells (BMECs).

Our results showed that EC-specific activation of miR-195 increased type H vessel number and stimulates new bone formation in aged mice.

In our study, we constructed EC-specific aptamers with secondary structures that are capable of molecular recognition specifically of endothelial cells, and combined them with angomiR-195 to specific elevate miR-195 level in endothelial cells.

qRT-PCR confirmed that intravenous injection of aptamer-agomiR-195 significantly increased the levels of miR-195 in ECs (Supplementary Fig. 7d).

Taken together, these results suggested that EC-specific activation of miR-195 by intravenous injection of aptamer-agomiR-195 promoted the CD31 [hi]Emcn [hi] vessel formation, and stimulated new bone formation in aged mice.

11

miR-150 regulates MNC migration by targeting Cxcr4 Since miR microarray analysis revealed that miR-29c, miR-98/let-7 family, miR-150, miR-195 and miR-494 expression was significantly downregulated in BM-derived MNCs, we extensively examined databases for predicted targets of these miRNAs involved in MNC mobilization.

Since miR microarray analysis revealed that miR-29c, miR-98/let-7 family, miR-150, miR-195 and miR-494 expression was significantly downregulated in BM-derived MNCs, we extensively examined databases for predicted targets of these miRNAs involved in MNC mobilization.

12

Expression of miR-365, miR-195, miR-30c and miR-200a were significantly downregulated in RRV infected cholangiocytes.

After 24 hours of RRV infection (100 multiplicity of infection [MOI]), the expression levels were downregulated by 1.30 to 1.53-fold for miR-365, miR-195, miR-30c and miR-200a (Figure  6) below levels of the vehicle control.

Among these, miR-30b/c, -195 and −365 had 1 [st] tier links to biological processes of hematology and inflammation by influencing the expression of Pim1, Il10ra, Il7r (miR-195), Arrb2 (miR-365), Pik3cd, Cmpk2, Socs3, Cysltr1, Serpine1 and Ceacam1 (miR-30b/c).

Focusing first on the pattern of postnatal development in the liver, miR-30c and -200a increased in suckling mice at all time points by 2–12 fold above adult levels (Figure  4, blue lines), while miR-195 tended to have lower levels but did not reach statistical significance.

05mmu-miR-365−4.620.015−4.450.034mmu-miR-133a−3.650.015−2.670.032mmu-miR-200b−3.960.015−2.840.046mmu-miR-133b−4.410.009−4.350.032mmu-miR-200a−5.560.009−3.110.034 mmu-miR-195 −4.93 0.014 −8.71 0.05 * Minus sign implies fold change below controls.

13

Notably, several large miRNA families (such as the miRNA-15, miRNA-30, and let-7 families) were upregulated in P10 cardiac ventricles, and miRNA-195 (a member of the miRNA-15 family) was shown to be the most highly upregulated miRNA.

Cardiomyocyte -targeted overexpression of miRNA-195 was associated with cardiac developmental defects and reduced cardiomyocyte proliferation in P1 mice (35).

In addition, cardiomyocyte-directed overexpression of miRNA-195 prevented cardiac regeneration of the neonatal mouse heart after induction of MI at P1.

14

According to previous studies in cancer (MCF-7) cells EGCG up-regulates the expression of miR-16, a member of the miR-15b family (family of miR-16/miR-15a/miR-497/miR-322/miR-195) and consequently, EGCG down-regulates Bcl-2 expression level and thus counteracts cancer progression [25].

A. Cartoon showing the murine mmu-miR-15b (family of miR-16/miR-15a/miR-497/miR-322/miR-195) with STIM2 3’-untranslated region (3’-UTR) with seed sequence.

15

P53 signaling pathway was regulated by ssc-miR-20b, ssc-miR-497 and ssc-miR-195 through targeting CCNG2, CDKN1A, CASP8, GADD45G, CHEK1, SESN1 and CCNE1.

Cell cycle and Neurotrophin signaling pathway were regulated by ssc-miR-20b, ssc-miR-128, ssc-miR-497, ssc-miR-195 and ssc-miR-371-5p through corresponding putative target genes.

Ssc-miR-195 and ssc-miR-497 were highly expressed in hpiPSCs and they were also located in the same genome loci in chromosome 12.

Ssc-miR-106a, ssc-miR-363, ssc-miR-195, ssc-miR-497, ssc-miR-146b, ssc-miR-92b-5p, ssc-miR-20b and ssc-miR-935 were highly expressed in hpiPSCs than that in mpiPSCs (Fig 3A).

16

For example, the MyomiRs miR-208a, -208b and -499 control myosin heavy chain isoform expression [4], miR-133a and miR-1 are crucial regulators of cardiac differentiation and development [1] and miR-195 overexpression is sufficient to induce hypertrophy in mice, while ablation of miR-208a is protective [5].

Three notable examples include: miR-499, a MyomiR that controls myosin heavy chain isoform expression [4] and is a biomarker of myocardial infarction [40]; miR-21, which is currently touted as a promising therapeutic target for cardiovascular diseases [41]; and miR-195, whose deletion in mice leads to cardiac hypertrophy [5].

17

Based on this observation, we combined the analysis from the TF gene coexpression network of TF genes and miRNAs and hypothesized that the functions related to cardiac hypertrophy were induced by the up-regulated TF genes in Module 1 at days 3 and 5 but suppressed by some miRNAs after day 7. Runt related transcription factor 1 (Runx1), for example, in Module 1 is a key regulator of heart post-myocardial infarction [24] and was up-regulated from day 3 to day 14 and likely suppressed by some known cardiac miRNAs, such as miR-126–5p, miR-195, miR-208b, and miR-21 at day 28 (Suppl.

18

Notably, 23 circulating miRNAs (mmu-miR-16, mmu-let-7i, mmu-miR-26a, mmu-miR-17, mmu-miR-107, mmu-miR-195, mmu-miR-20a, mmu-miR-25, mmu-miR-15b, mmu-miR-15a, mmu-let-7b, mmu-let-7a, mmu-let-7c, mmu-miR-103, mmu-let-7f, mmu-miR-106a, mmu-miR-106b, mmu-miR-93, mmu-miR-23b, mmu-miR-21, mmu-miR-30b, mmu-miR-221, and mmu-miR-19b) were significantly downregulated in DIO mice but upregulated in DIO + LFD mice.

As shown in the Venn diagram in Fig.   7, notably, 23 of the 28 upregulated miRNAs in DIO + LFD mice (mmu-miR-16, mmu-let-7i, mmu-miR-26a, mmu-miR-17, mmu-miR-107, mmu-miR-195, mmu-miR-20a, mmu-miR-25, mmu-miR-15b, mmu-miR-15a, mmu-let-7b, mmu-let-7a, mmu-let-7c, mmu-miR-103, mmu-let-7f, mmu-miR-106a, mmu-miR-106b, mmu-miR-93, mmu-miR-23b, mmu-miR-21, mmu-miR-30b, mmu-miR-221, and mmu-miR-19b) were downregulated in the DIO mice.

19

Interestingly, we also found these two confirmed BDNF -targeting miRNAs (miR-30a and miR-195) were significantly up-regulated in mouse brain and liver after RDX exposure (Tables 1 and 2, Figure 4).

Of the 113 miRNAs with significantly aberrant expressions after RDX exposure, the expression levels of 10 miRNAs were significantly increased in both mouse liver and brain (p < 0.01): miR-99a, miR-30a, miR-30d, miR-30e, miR-22, miR-194, miR-195, miR-15a, miR-139-5p, and miR-101b.

In that study, luciferase assays confirmed that BDNF was targeted by two miRNAs, miR-30a-5p and miR-195 (Mellios et al. 2008).

20

Similarly, miR-195 (predicted to target BDNF), and let-7b and miR-98 (both predicted to target NGF) have been shown previously in human muscle to increase with aging [45], consistent with our analysis in VO rat muscle.

org) revealed several target sites for BDNF: miR-206-3p, miR-10a-5p, miR-1b, miR-195-5p, and miR-497-5p that were conserved in humans.

In VO rat muscle, miR-206-3p, miR-195-5p, and miR-497-5p were increased significantly.

MicroRNAs predicted to influence neurotrophins: a BDNF (miR-206-3p, miR-10a-5p, miR-1b, miR-195-5p and miR-497-5p), b NT3 (miR-21-5p, miR-222-3p and miR-221-3p), and c NGF (let-7b-5p and miR-98-5p) were quantified by qPCR analysis in YA (n = 8) vs VO (n = 10) rat vastus lateralis muscle and WT (n = 8) vs Sarco (n = 7) gastrocnemius muscle.

In contrast to the aforementioned studies in aging muscle, most of the miRNAs studied herein have been examined in the context of experimental denervation, including miR-206 (increases after reinnervation), miR-10a-5p (increases four- to seven-fold with denervation), miR-1 (increases up to 10-fold following denervation and remains elevated after reinnervation), miR-195 (increases up to 10-fold with denervation), miR-21 (increases with denervation), miR-221 (no consistent change), miR-222 (no consistent change), and miR-98 (increases up to 10-fold with denervation) [43, 47, 48].

For BDNF, our analysis identified increases in miR-206-3p, miR-195-5p, and miR-497-5p in VO rat muscle.

21

However, the transcription factor Myb was downregulated with concurrent overexpression of miR-15b, miR-16, miR-150 and miR-195 (Figure 3 and Additional file 3).

Many miRNAs that we identified as being differentially expressed in granulopoiesis were not previously implicated in this process including miR-19a, miR-19b miR-24, miR-26a, miR-26b, miR-93, miR-106b, miR-191, miR-139-5p, miR-140 and miR-195 (Figures 2A and 3, Additional file 1).

Amongst these are those differentially regulated in our study including miR-16, miR-19a, miR-26a, miR-26b, miR-139, miR-195 and miR-223.

22

The screening predicted five breast cancer-related miRNAs (miR-17, miR-25, miR-124, miR-195, and miR-340) to target MCU.

There are significant differences between high and low metastatic breast cancer cell lines in the expression levels of miR-124, miR-195 or miR-340 (Figure 3A-3E).

Figure 3 (A-E) Expression of miR-17, miR-25, miR-124, miR-195 and miR-340 in a panel of four breast cancer cell lines by quantitative polymerase chain reaction analysis.

Previous studies demonstrated several deregulated miRNAs may promote breast cancer proliferation and migration, including miR-340, miR-195 and miR-124 [21– 23].

23

Decreased expression of miR-298 and miR-195 is predicted to result in increased protein expression of Myf5 and Smad2, respectively, in emerin -null myogenic progenitors.

Thus, reduction of miR-298 and miR-195 results in increased expression of their predicted targets.

24

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).

The analysis showed miRNAs that were related to ER stress pathway (let-7f, miR-351, miR-127, miR-133a, miR-195, miR-214 and miR-503), suggesting CASP3, CASP7, XBP1, ATF6 and ATF4 as possible target genes for these miRNAs (Table 4).

Hcy also induces alteration of miRNAs related to tight junctions signaling such as miR-128, miR-132, miR-133, miR-195, miR-3473, miR-19, miR-200, miR-205, miR-214, miR-217, miR-23, miR-26, miR-29, miR-30, miR-31 AND miR-690.

25

Furthermore, miRNAs were confirmed to be upregulated upon myelination: p≤0.0001 (miR-34a, miR-146b), p = 0.04 (miR-338-3p), p = 0.003 (miR-204), p = 0.0007 (miR-27b), p = 0.005 (miR-140), p = 0.0002 (miR-138), p = 0.01 (miR-195), p = 0.0004 (miR-30a).

All miRNAs analyzed were significantly downregulated in Dicer [fl/fl] Dhh-Cre [+] nerves at p4: p≤0.0001 (miR-34a, miR-146b, miR-338-3p, miR-204, miR-27b, miR-140, miR-138, miR-30a), p = 0.0002 (miR-195).

However, some miRNAs identified have not been previously reported to be involved in myelination, including miR-195, miR-140, miR-34a, miR-30a, miR-30b, miR-30c, and miR-140*.

26

miRNA expression fold change was determined by the ΔΔCT method, using the geometric mean of miR-10a and miR-195 to normalize data.

All data were normalised to the expression of two reference microRNAs (mmu-miR-10a-5p and mmu-miR-195a-5p) that we found to be invariable and ubiquitous endogenous in the Taqman PCR array data set and then validated by the individual Taqman qPCR assays in the extended cohort (S2 Table).

Mean dCt were derived from the geometric mean of mmu-miR-10a-5p and mmu-miR-195-5p (the least variable microRNAs across all samples).

The (A) geometric mean of mmu-miR-10a-5p and mmu-miR-195-5p or (B) mmu-miR-10a-5p alone was used as a reference microRNA (the least variable microRNA(s) across all samples in the present experimental setup).

Both miR-10a and miR-195 were determined to be invariable and ubiquitous endogenous controls by two approaches; cel-miR normalisation of array data (data not shown), and subsequent qPCR data (S2 Table).

27

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].

28

For example, microRNA-375, miR-29c, miR-195, miR-625, miR-203, miR-302b, miR-133a, miR-101, miR-27a, miR-655 and miR-200b can suppress the growth of ESCC cells by regulating the expression of a variety of molecules, including IGF1R (insulin-like growth factor 1 receptor), cyclin E, Cdc42, Sox2, Ran, ErbB4, FSCN1 and MMP14, enhancer of zeste homolog 2 (EZH2), KRAS, ZEB1, TGFBR2 and Kindlin-2. In this study, we revealed the inhibitory effects of both miR-26a and miR-144 on proliferation and metastasis of ESCC.

29

miR-195a-5p that has the same seeding sequence with miR-16-5p, is predicted to bind BCL2 mRNA, and this was also downregulated in our study and may increase further the anti-apoptotic effects of miR-21-5p and miR-16-5p.

miR-195a-5p that has the same seeding sequence with miR-16-5p and is predicted to bind BCL2 mRNA was also downregulated while miR-153-3p that was also found to bind BCL2 experimentally (by Western blot, qRT-PCR, and LUC) [45] was increased.

30

microRNA-195 suppresses angiogenesis and metastasis of hepatocellular carcinoma by inhibiting the expression of VEGF and CDC42 [19].

31

In naïve peripheral CD3 [+] T cells, we found six miRNAs exclusively up-regulated in DBA-1/J mice (miR-195, miR-689, miR-500, miR-196b, miR-10a and miR-805) and four exclusively up-regulated in DBA-2/J mice (miR-467e, miR-101a, miR-125-5p and miR-669a).

32

We confirmed that six of the eight selected down-regulated hsa-miRNAs (miR-145, miR-497, miR-150, miR-342-5p, miR-34b* and miR-100) were significantly down-regulated in NPC tissues, whereas miR-195 and miR-143 exhibited no significant difference between the two groups of subjects (Fig. 1D).

33

Moreover, a trend toward downregulation of miR-195 and 499 was observed in that study, where we found significant downregulation of both miRNAs after 3 days of T3 treatment.

34

MicroRNA-195 downregulates Alzheimer's disease amyloid-β production by targeting BACE1.

35

This confirms our hypothesis that α-syn can regulate PP2A methylation via LCMT-1 and PME-1. The expression of LCMT-1 and PME-1 have recently been found to be post-trancriptionally regulated by microRNA-195 (Liu et al., 2016a).

Knockdown of microRNA-195 contributes to protein phosphatase-2A inactivation in rats with chronic brain hypoperfusion.

36

By contrast, microRNA-208a and microRNA-195 were up-regulated in cardiac hypertrophy, which were sufficient to drive pathological cardiac growth when over-expressed in transgenic mice, respectively [10, 11].

37

miRNA 7-bp motif MBS index Match position Mature miRNA sequence* miR-489 TGGTGTC 1 4-10aat GACACCAcatatatggcagcmiR-666 [†] GCTGTGC 2 6-12agcgg GCACAGCtgtgagagccmiR-292-3p [†] ACCTGGC 8 8-14aagtgcc GCCAGGTtttgagtgtmiR-196b [†] ACAACAG 10 12-18taggtagtttc CTGTTGTtggmiR-685 [†] GTGCCTC 14 15-21tcaatggctgaggt GAGGCAC miR-195 CTGTGCT 16 5-11tagc AGCACAGaaatattggcmiR-330 [†] CTGTGCT 16 5-11gcaa AGCACAGggcctgcagaga miR-424 GAATTGC 27 6-12cagca GCAATTCatgttttgga miR-219 GAATTGC 27 14-20tgattgtccaaac GCAATTCtmiR-488* [†] ACAGCCT 30 6-12ttgaa AGGCTGTttcttggtc Table 5 MiRNAs that match with two predicted 7-bp motifs (one G-U pair allowed) miRNA 7-bp motif MBS index Match position Mature miRNA sequence* miR-195 GTGTTGC 6 3-9ta GCAGCACagaaatattggc CTGTGCT 16 5-11tagc AGCACAGaaatattggc miR-196b ACAACAG 10 12-18taggtagtttc CTGTTGTtgg AGGGAAC 21 7-13taggta GTTTCCTgttgttggmiR-330 [†] CTGTGCT 16 5-11gcaa AGCACAGggcctgcagaga GGTCCTG 18 9-15gcaaagca CAGGGCCtgcagagamiR-666 [†] GCTGTGC 2 6-12agcgg GCACAGCtgtgagagcc CTGTGCT 16 5-11agcg GGCACAGctgtgagagccmiR-710 [†] CAGGACT 19 4-10cca AGTCTTGgggagagttgag CTCTTCC 29 11-17ccaagtcttg GGGAGAGttgag Potential TFBS and MBS were selected based on the number of occurrences of 6-bp elements in 5'-proximal flanking region of transcription starting sites and 7-bp elements in 3'-untranslated regions of mRNA, respectively, as detailed in the methods.

miRNA 7-bp motif MBS index Match position Mature miRNA sequence* miR-489 TGGTGTC 1 4-10aat GACACCAcatatatggcagcmiR-666 [†] GCTGTGC 2 6-12agcgg GCACAGCtgtgagagccmiR-292-3p [†] ACCTGGC 8 8-14aagtgcc GCCAGGTtttgagtgtmiR-196b [†] ACAACAG 10 12-18taggtagtttc CTGTTGTtggmiR-685 [†] GTGCCTC 14 15-21tcaatggctgaggt GAGGCAC miR-195 CTGTGCT 16 5-11tagc AGCACAGaaatattggcmiR-330 [†] CTGTGCT 16 5-11gcaa AGCACAGggcctgcagaga miR-424 GAATTGC 27 6-12cagca GCAATTCatgttttgga miR-219 GAATTGC 27 14-20tgattgtccaaac GCAATTCtmiR-488* [†] ACAGCCT 30 6-12ttgaa AGGCTGTttcttggtc Table 5 MiRNAs that match with two predicted 7-bp motifs (one G-U pair allowed) miRNA 7-bp motif MBS index Match position Mature miRNA sequence* miR-195 GTGTTGC 6 3-9ta GCAGCACagaaatattggc CTGTGCT 16 5-11tagc AGCACAGaaatattggc miR-196b ACAACAG 10 12-18taggtagtttc CTGTTGTtgg AGGGAAC 21 7-13taggta GTTTCCTgttgttggmiR-330 [†] CTGTGCT 16 5-11gcaa AGCACAGggcctgcagaga GGTCCTG 18 9-15gcaaagca CAGGGCCtgcagagamiR-666 [†] GCTGTGC 2 6-12agcgg GCACAGCtgtgagagcc CTGTGCT 16 5-11agcg GGCACAGctgtgagagccmiR-710 [†] CAGGACT 19 4-10cca AGTCTTGgggagagttgag CTCTTCC 29 11-17ccaagtcttg GGGAGAGttgag In this study, we extended the application of MotifMo deler to simultaneously identify putative cis-acting elements that represent both transcription factor and miRNA binding sites from array-derived gene expression data.

38

For example, Landgraf et al. [18] studied the mammalian miRNA expression atlas by sequencing 250 small RNA libraries representing 26 different human and rodent organ systems and cell types, and Wang et al. [21] used home-made miRNA microarrays to identify the rat lung-specific miRNAs, miR-195 and miR-200c.

Wang et al. [21] investigated the tissue-specific expression of miRNAs in six rat tissues (lung, heart, brain, kidney, liver and spleen), and found that miR-195 and miR-200c were expressed specifically in the lung.

39

We have previously shown that miR-195 downregulation in HCC leads to increased VAV2 expression, which in turn stimulates the Rac1 signaling and thereby promotes metastasis of HCC cells in vitro and in vivo [29].

40

Furthermore, overexpression of miR-195 in human MSCs reduced osteogenic differentiation and enhanced cell proliferation through regulation of VEGF protein translation [26].

41

For example, miR-195 expression is reduced in tumor tissues and associated with poor survival outcomes [18].

For example, miR-195 expression is low in tumor tissues and associated with the clinical stages of NSCLC [18].

42

Similarly, miR-29, miR-195 and miR-208 were dramatically reduced upon MI, but were partially restored by overexpression of miR-24 in cardiomyocytes (Fig. 4K).

We performed qPCR to detecct the expression levels of ANF, BNP and several stress-responsive miRNAs (miR-29, miR-195 and miR-208) in Myh6-miR-24 transgenic and control hearts.

43

There are previous reports of the involvement of miRNAs in retinal development, function, and photoreceptors survival 1, 2 and also the involvement of different miRNAs in the pathogenesis of various diseases of the retina including miR-9, miR-34a, miR-125b, and miR-155 in macular degeneration, 17, 18 miR-146a and miR-195 in diabetic retinopathy, 19, 20 and miR-125a and miR-17 in retinoblastoma.

Mortuza R, Feng B, Chakrabarti S. miR-195 regulates SIRT1 -mediated changes in diabetic retinopathy.

44

In addition, prefrontal cortical miRNA expression levels have been reported to be altered in other psychiatric disorders: for instance, let-7d was shown to be up-regulated in the PFC of spontaneous hyperactive rats, an animal mo del for attention deficit hyperactivity disorder (ADHD) (35), and miR-195 was demonstrated to fine-tune regional levels of brain derived neurotrophic factor (BDNF) in the PFC of schizophrenic patients (36).

45

Of the eight differentially expressed miRNAs found in both males and females exposed to O [3], a total of six miRNAs were upregulated exclusively in males: miR-338-5p (log fold change = 1.636), miR-222-3p (log fold change = 0.699), miR-130b-3p (log fold change = 0.646), let-7i-5p (log fold change = 0.552), miR-195a-5p (log fold change = 0.543), and miR-144-3p (log fold change = 0.427) (Fig.   2).

46

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].

47

A recent study demonstrated that the miR-195/497 cluster can suppress cell proliferation and invasion of breast cancer [34].

Our results indicate that the miR-30 family may function together with the let-7 family and the miR-195/497 cluster to control cell proliferation by cooperatively destroying the Ras signaling pathway.

The sub-network II is composed of six members of the let-7 family (let-7a, let-7b, let-7c, let-7d, let-7f, and let-7g), the miR-30 family, the miR-195/miR-497 cluster, and two unrelated miRNAs including miR-26b and miR-150.

48

Hang P Sun C Guo J Zhao J Du Z BDNF-mediates Down-regulation of MicroRNA-195 Inhibits Ischemic Cardiac Apoptosis in RatsInt J Biol Sci.

49

The miR-15 family members including miR-15a, miR-15b, miR-16, miR-195, miR-424, and miR-497, show 5′-end sequence similarity and many common targets [16, 17].

It has been demonstrated that miR-195 is pro-apoptotic in cardiomyocytes [20].

It was reported miR-195 increases cardiac hypertrophy [27] and worsens systolic dysfunction in mice with MI [17], while miR-15b, another member of the same miR-15 family, was found to attenuate myocardial fibrosis and hypertrophy in pressure-overloaded mice [19].

50

2014.282 25174400 6. Cai C miR-195 inhibits tumor progression by targeting RPS6KB1 in human prostate cancerClin.

51

For example, Ccnd1 is targeted by miR-138, but it is also targeted by miR-34a, miR-16, miR-195, miR-153, miR-503 and many other microRNAs [50].

52

Importantly, also, miR-497 and miR-15b, which target the same seed region of miR-16, were found among the top candidates (cluster #1, Table  2), and the other miRNAs belonging to the same family (miR-195, miR-15a, and miR-424), even if less efficiently, all showed an inhibitory effect on A549 cells (0.902, 0.837, and 0.834 normalized A549 cell number, respectively).

53

Liu C, Guan H, Wang Y, Chen M, Xu B, Zhang L, Lu K, Tao T, Zhang X, Huang Y. miR-195 inhibits EMT by targeting FGF2 in prostate Cancer cells.

54

[31] Overexpression of miR-101 in HCC mice mo del reduces epithelial–mesenchymal transition (EMT) and angiogenesis by repressing EZH2, COX2, STMN1 and ROCK2 [22] and miR-195 suppresses angiogenesis by reducing VEGF, VAV2 and CDC42.

55

As in the case of miR-199a, miR-195 also targets esrrg.

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.

56

Improved reprogramming efficiency has been achieved by the knocking-down expression of senescence -associated proteins p53, p21 [CIP1], p16 [INK4A] and p14 [ARF] 3, 10, 11, and microRNA-195 [12].

57

Also miR-195 was upregulated in the serum of breast cancer patients in the study by Mishra et al. [82], and served within a signature for early detection.

58

Other differentially expressed miRNAs specific to the mouse mo del were also validated by qPCR, including miR-195 (member of the miR-15 family) and miR-541 family members.

25E-0316mmu-miR-15a-5pmir-150.209.185.63E-052.24E-0320mmu-miR-195-5pmir-150.248.631.26E-044.01E-0353mmu-miR-15b-5pmir-150.156.854.15E-034.99E-0244mmu-miR-93-5pmir-170.166.981.73E-032.45E-0221mmu-miR-181d-5pmir-1810.248.471.51E-044.59E-0357mmu-miR-181a-5pmir-1810.209.815.96E-036.44E-0269mmu-miR-182-5pmir-1820.628.718.90E-038.21E-0262mmu-miR-183-5pmir-1830.668.386.90E-036.

59

qRT-PCRwas conducted for miRs that had been previously described to be involved in pathological cardiac hypertrophy(miR-21 and miR-195) and in mo dels of cardiovascular disease associated with cardiomyocyte injury (miR-499).

In particular, we did not find altered levels of miR-195, -499, -341*, -328, -680 and -1224.

60

Additionally, we have shown that miR-195 regulates SIRT1 expression 27.

61

Moreover, miR‐101a‐5p and miR195a‐5p that were downregulated by CR in young mice have been reported to cause cardiac fibrosis via TGFβ pathway (Pan et al., 2012; Tijsen et al., 2014).

62

Interestingly, among the miRNAs found to be upregulated in exosomes in response to cytokines, several of them including miR-146a, miR-146b, miR-195, miR-290a-3p, miR-362-3p and miR-497 are known to be involved in cell death [29- 34].

63

The results in this study and previously completed human studies reveal that the expression level of miR-195, 222, 200c, 21, and let-7d was higher in high graded fibrotic liver tissue than in low graded fibrotic liver tissue.

64

Long et al found that circulating miR-30a and miR-195 were highly expressed while let-7b was significantly lower in AMI patients at 8 h after onset of AMI and suggested that the plasma concentration of miR-30a, miR-195 and let-7b could be potential indicators for AMI [48].

65

To date, mRNAs and microRNAs associated with HCC development are innumerably reported, among which mRNAs and microRNAs pro- or anti- metastasis and angiogenesis remain highlights in recent several years, such as twist1 [24], CXCR6 [25], vasohibin2 [26], mir-29b [6], mir-195 [12].

To date, relatively few studies have reported that miRNAs possess both metastasis and angiogenesis functions in HCC, such as mir-29b [11], mir-195 [12].

66

Four miRs, representing the most highly expressed members of this family (miR-16, miR 103 and miR-195), were present in the array used in our study.

67

Chen YQ Abated microRNA-195 expression protected mesangial cells from apoptosis in early diabetic renal injury in miceJ.

68

On the other hand RNA -based therapies are attractive due to their specificity [60] The list of altered miRs in chronic diabetic complications include miR-200b, miR-155, miR-1, miR-320, miR-195 among others [28, 43, 60, 61]; each targeting various RNA molecules.

69

Seven miRNAs (miR-21, miR-30a, miR-126, miR-133a, miR-195, miR-208a and miR-320a) were confirmed to be differentially expressed between CAD and control samples (Fig. 1B).

70

Systemic miRNA-195 differentiates breast cancer from other malignancies and is a potential biomarker for detecting noninvasive and early stage disease.

71

For example, miRNA-21, miRNA-23a, miRNA-24, miRNA-133, miRNA-208/miRNA-195 and miRNA-199 have been shown to be involved in cardiac hypertrophy (15- 17), miRNA-1 in arrhythmia (18), miRNA-29 and miRNA-21 in cardiac fibrosis (19, 20), miRNA-210 and miRNA-494 in ischemic heart disease (21) and miRNA-129 in heart failure (22).

72

283A impairs the proper RNA processing machinery of pri-miR-195 and reduces the production of mature miR-195, which in turn leads to the derepression of many miR-195 targets related to cell proliferation 13.

73

The remaining four miRNAs were either not detected in this new set of mice (miR-335-5p) or did not show a significant change of the expression levels (miR-140-5p, miR-195a-5p and miR-140-3p) (Fig.   2A).

74

For instance, miR-195 targets a cadre of extracellular matrix proteins and displays significant inverse correlation with aortic diameter [23].

75

Overexpression of miR195 and -199a in cardiomyocytes also provoked myocyte enlargement [11].

76

For example, a DE microRNA miR-195a-5p that was up-regulated in the WI sample compared to the sham sample, is associated with transmembrane potential of mitochondria and mitochondrial membrane [24], indicating ischemia likely starts to impair the function of mitochondria in liver cells.

77

A number of further targets were identified through miRNA/protein correlations, such as the mir-200 family (miR-200a, miR-200b and miR-200c), miR-191, miR-195, miR-301, and miR-322 (Figure 4D).

78

Several miRNAs, such as miR-101 [6], miR-122 [7], [8], [9] miR-373 [10], miR-221/222 [11], [12], [13], miR-195 [14], miR-30d [15], miR-125b [16], miR-18a [17], miR-139 [18], miR-223 [19] and miR-29 [20], have already been reported to regulate HCC tumor progression and metastasis by regulating key genes such as Mcl-1, ADAM17, YAP, DDIT4, Cyclin D1, CDK6, E2F3, Galphai2, LIN28B, estrogen receptor-α, Rho-kinase 2, Stathmin 1 and Bcl-2 and so on.

79

Systemic miRNA-195 differentiates breast cancer from other malignancies and is a potential biomarker for detecting noninvasive and early stage disease.

80

A recent study demonstrated that high-fat diet feeding after five months induced a different miRNA expression pattern in the heart, identifying miR-21a-3p, miR-29c-3p, and miR-195a-3p related to the glucose metabolic pathway [19].

81

The exceptions were miRNAs miR-16, miR-195 and miR-197, which could be detected despite.

The exceptions were miRNAs including miR-16, miR-195 and miR-197, which were previously shown to be RNase-resistant (Figure 1B, [35]).

82

We also observed the suppression of miRNAs in SCC samples compared to uninvolved mice skin for miR-195-5p, miR-127-3p, miR-206-3p, miR-411-5p, miR-709-5p, and miR-322-5p.

83

In addition, CCND2 can be regulated by miR-206 [25], miR-340 [26], miR-154 [27], miR-133a [28], miR-155 [29], miR-1 [30], miR-16 [31], miR-610 [32], miR-26a [33], miR-195 [34], and miR-204 [35], which have roles in cellular proliferation disorders (e. g., breast cancer, cervical carcinoma, and retinoblastoma).

84

MiR-195 may promote cardiomyocyte apoptosis by targeting Bcl-2 and inducing mitochondrial apoptotic pathway during ischemia reperfusion -induced myocardial injury [27].

85

Previous studies have demonstrated that several miRNAs are involved in the regulation of HCC angiogenesis, for example, miR-195, miR-491, miR-126-3p, etc [20– 22].

86

283+A was shown to regulate miR-195 maturation by base pairing to pri-miR-195 and blocking its processing by Drosha 22.

87

These include miRNAs with known roles in breast cancer development such as miR-7, miR-10b, miR-21, miR-100, miR-155, miR-195, miR-221, and miR-218.

88

MicroRNA-195 targets ADP-ribosylation factor-like protein 2 to induce apoptosis in human embryonic stem cell-derived neural progenitor cells.

89

Soon P. S. Tacon L. J. Gill A. J. Bambach C. P. Sywak M. S. Campbell P. R. Yeh M. W. Wong S. G. Clifton-Bligh R. J. Robinson B. G. miR-195 and miR-483-5p Identified as Predictors of Poor Prognosis in Adrenocortical CancerClin.

Chabre O. Libé R. Assie G. Barreau O. Bertherat J. Bertagna X. Feige J. J. Cherradi N. Serum miR-483-5p and miR-195 are predictive of recurrence risk in adrenocortical cancer patientsEndocr.

90

In all, we found moderate (three-fold) region-specific enrichment of 48 miRNAs (e. g. miR-138, miR-195 and miR-218, Figure S2).

In line with previous studies in mice, we also found moderate enrichment of miR-195 [39], [40] in the cerebellum and of miR-218 in the hippocampus [39] (Figure S2).

91

Numerous studies have already shown the involvement of microRNAs in AAA development, including miR-195, miR-21, miR-29b, and miR-24 [19– 22].

92

with and without species identifiers (e. g. hsa-miR-195 and miR-195).

93

This ncRNA-ncRNA interaction prevents pri-miRNA-195 cleavage by Drosha.

94

Among these miRNAs, miR-142-5p [26], miR-223 [27], miR-22 [28, 29] miR-24 [17], miR-497 [30], and miR-195 [30] have been found to participate fracture healing.

95

Zheng and colleagues reported that the silencing of miR-195 reduced lung and liver injuries and improved the survival of septic mice (88).

96

At 15 dpi, 17 miRNAs (let-7b,c, miR-10a, miR-21, miR-25, miR-26a, miR-29c, miR-30a,b,c,d, miR-99a, miR-103, miR-151, miR-195, and miR-200b,c) were identified to be important in the late repair phase.

97

miR-107, together with miR-15a/b, miR-16, miR-103, miR-195, miR-424, miR-497, miR-503, and miR-646, belongs to the miR-15-107 group.

98

Real-time PCR was performed to investigate the expression of mmu-miR-1907, mmu-miR-15a, mmu-miR-15b, mmu-miR-497, mmu-miR-16, mmu-miR-322 and mmu-miR-195 in the brain tissue of F1 mice from the two groups.

99

283+A was shown to decrease pri-miR-195 biogenesis by impairing microprocessor recognition and pri-miRNA cropping [16].

100

In mice, on the other hand, Bak et al. reported that miR-195, miR-497, and miR-30b are enriched in the mouse cerebellum (Bak et al., 2008) whereas Hohjoh and Fukushima reported cerebellar enrichment of miR-16, - miR-34a, in addition to miR-195 (Hohjoh and Fukushima, 2007).