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9 publications mentioning dre-mir-142a

Open access articles that are associated with the species Danio rerio and mention the gene name mir-142a. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1
[+] score: 345
We show that Cdh5 is a potential target of miR-142a-3p and demonstrate that vascular integrity, remo deling and angiogenesis can be influenced through modulation of miR-142a-3p expression, making this a potentially important target for exploring either pro or anti – angiogenesis therapies. [score:7]
We also analyzed the endogenous levels of cdh5 transcript in zebrafish embryos upon overexpression and downregulation of miR-142a-3p to investigate the direct modulation of cdh5 expression by miR-142a-3p (Figure 7). [score:7]
Zebrafish embryos sequentially injected with GFP- cdh53’UTR mRNA and miR-142a-3p duplex showed suppression of GFP expression, while miR-144 duplex had no effect on the GFP expression (Figure 6B). [score:7]
First, in order to check the endogenous expression of miR-142a-3p in blood vessel, we examined the relative expression levels of miR-142a-3p in FAC sorted endothelial cells expressing GFP under the promoter of fli1 transcription factor using 2dpf Tg (fli1:EGFP, gata1a: dsRed) developing embryo. [score:7]
C - cdh5 relative expression quantified by QRT–PCR upon downregulation of miR-142a-3p. [score:6]
In summary we show that miR-142a-3p directly binds with the two predicted two target sites in the 3′UTR of cdh5 gene and modulates the expression of cdh5 transcript and protein in zebrafish embryos. [score:6]
In parallel, down regulation of the endogenous miR-142a-3p resulted in increased expression of endogenous cdh5 gene, suggesting a direct interaction between miR-142a-3p and cdh5 gene to regulate vascular integrity and remo deling in developing zebrafish embryos. [score:6]
So we hypothesized that, if lmo2 gene regulates expression of cdh5/miR-142 transcriptionally and miR-142a-3p regulates cdh5 gene post-transcriptionally, then lmo2, cdh5 and miR-142a-3p may be part of an interacting network that could regulate angiogenesis and vasculature remo deling in zebrafish embryos. [score:6]
Since, it is well documented that miRNAs can regulate multiple genes, the observed vascular phenotype could be a result of miR-142a-3p targeting cdh5 along with other bonafied target genes. [score:6]
During zebrafish embryo development, miR-1 is known to express ubiquitously where as miR-144 and miR-142a-3p show strong expression in blood cells [35]. [score:6]
We found that expression of cdh5 increased by approximately 1.58±0.32 fold upon morpholino -mediated downregulation of endogenous miR-142a-3p (Figure 7C). [score:6]
Studies conducted on human cancers by other groups from have indicated that LMO2, a transcription factor expressed in blood and vasculature acts as a transcriptional suppressor of miR-142 [32]. [score:5]
Strong miR-142a-3p induced suppression of GFP expression was observed with both the predicted binding sites present in the 3′UTR of cdh5 (Figure 6C). [score:5]
Co-injection of the miR-142a-3p and GFP-4X PT 3′UTR mRNA led to suppression of GFP expression in wildtype zebrafish embryos. [score:5]
In the presence of high levels (overexpression) of miR-142a-3p, cdh5 levels are suppressed (Figure 7A and 7B). [score:5]
B - Co-injection of the miR-142a-3p and GFP- cdh5 3′UTR mRNA leads to suppression of GFP expression in wildtype zebrafish embryos. [score:5]
A - cdh5 relative expression quantified by QRT–PCR upon overexpression of miR-142a-3p. [score:5]
Co-injection of the GFP- vegfab 3′UTR mRNA and miR-142a-3p led to moderate suppression of GFP expression in wildtype zebrafish embryos. [score:5]
Strong miR-142a-3p induced suppression of GFP expression was observed with both the predicted binding sites (GFP-3′UTR 3X site A and GFP-3′UTR 3X site B) [. ] [score:5]
Next we checked the levels of endogenous cdh5 transcript upon downregulation of endogenous miR-142a-3p in zebrafish embryos. [score:4]
In the absence (downregulation) of miR-142a-3p, the repressive influence of the miR-142a-3p on cdh5 is relieved, resulting in abnormal vascular remo deling (Figure 5B –5G, S1 and 7C). [score:4]
We argued that if the hemorrhage and vessel remo deling is specifically caused by over expression of miR-142a-3p then the down regulation of the endogenous levels of miR-142a-3p should also cause a phenotype in the same tissue i. e. blood vessel. [score:4]
Recently, it has been shown that inhibition of miR-142-3p specifically resulted in abnormal cardiac development in developing zebrafish embryos [38]. [score:4]
We observed that miR-142a-3p was ∼ 22.5 fold over expressed in GFP positive cells compared to GFP negative cells suggesting its enriched expression in endothelial cells (Figure 5A). [score:4]
We speculate that endogenous miR-142-3p could have other targets that may be involved in vascular development. [score:4]
The miR-142a-3p MO injected animals at 200 µM has displayed reduced expression level of endogenous miR-142a-3p to 0.01±0.007 (Figure 5C) In the miR-142a-3p knockdown embryos (28–30 hpf), the primary vasculature appeared normal, however the intersegmental vessels (Se) displayed defects and abnormal remo deling in a significant proportion (≥78%) of the injected embryos (Figure 5D –5H, Figure S1B – S1G). [score:4]
VE-cadherin (cdh5) an endothelial-specific transmembrane component of the adherens junction complex and an important regulator of endothelial cell-cell adhesion, serves as a target for repression by miR-142a-3p. [score:4]
No suppression of GFP expression was observed in zebrafish embryos in the sensor assay when 3′UTR of vegfab gene was co -injected with the miR-142a-3p duplex (Figure S3B). [score:4]
Our study also revealed a minor proportion of animals with impaired cardiac development upon suppression of cdh5 by miR-142a-3p. [score:4]
I - miR-142a-3p relative expression quantified by QRT–PCR in Lmo2 knockdown zebrafish embryos. [score:4]
The relative expression of miR-142a-3p was normalized to miR-26a [44]. [score:3]
Overexpression of miR-142a-3p in developing zebrafish embryos resulted in significant reduction of endogenous cdh5 gene at transcript as well as protein level. [score:3]
Taken together we speculate that lmo2, in addition to its known role as a transcriptional regulator of CDH5 and miR-142, may also regulates cdh5 post-transcriptionally through miR-142a-3p in zebrafish embryos. [score:3]
The specificity of the miR-142a-3p duplex induced hemorrhage phenotype was tested by injecting an antisense morpholino targeting the mature form of miR-142a-3p. [score:3]
The bioactivity of GFP-Sensor with 4X miR-142a-3p perfect complimentary target (PT) sites and miRNA duplex is shown in Supplementary Figure S5. [score:3]
We show that miR-142a-3p interacts with VE-cadherin (cdh5) gene in zebrafish by targeting the binding sites in the 3′UTR of cdh5 mRNA. [score:3]
Overexpression of miR-142a-3p in zebrafish embryos induces vascular defects. [score:3]
This effect is rescued, in part by the suppression of miR-142a-3p, restoring proper vascular integrity and remo deling (Figure S2). [score:3]
Ectopic overexpression of miR-142a-3p duplex in Tg (fli1:EGFP, gata1a: dsRed) zebrafish embryos resulted in hemorrhage in head and trunk region, and cranial vessel remo deling in 45–55% of the injected embryos at 2 dpf (Figure 3C ). [score:3]
Animals with impaired lmo2 function also displayed an increase in miR-142a-3p transcript level and a corresponding decrease in the expression of cdh5 transcript and protein (Figure 8G–8I). [score:3]
In our study we observe that suppression of the repressive influence of miR-142a-3p on cdh5 also results in higher levels of VE-Cad, leading to abnormal remo deling in the intersegmental vessel (Se) (Figure 5B –5G, 7C and S1B – S1G). [score:3]
In the present study we have tested only vegfab and cdh5 (Figure 6, 7 and S3) and we show that cdh5 gene is one of the bonafied targets of miR-142a-3p in zebarfish embryo. [score:3]
The relative expression of miR-142a-3p was normalized to miR-26a. [score:3]
Collectively, these data suggest that miR-142a-3p targets the 3′UTR of cdh5 gene via the two predicted recognition sites (Figure 6D). [score:3]
Figure S5 Bioactivity of GFP sensor with 4× miR-142a-3p perfect complimentary target (PT) sites and miRNA duplex. [score:3]
B - Silencing effect of miR-142a-3p on the GFP- vegfab 3′UTR gene target. [score:3]
Previously we noticed that ectopic overexpression of miR-142a-3p duplex in zebrafish embryos caused hemorrhage in head and trunk region and abnormal cranial vessel remo deling. [score:3]
Quantitative real-time PCR analysis in the single MO injected embryos revealed that the expression of cdh5 decreased by approximately 1.78 fold (0.56±0.05) compared to control and the miR-142a-3p expression increased by approximately 1.39±0.14 fold compared to control (Figure 8G and 8I). [score:3]
Overexpression of miR-142a-3p Induces Cerebral/trunk Hemorrhage and Vascular Remo deling in Zebrafish Embryos. [score:3]
B - Schematic of miR-142a-3p perfect target (PT) sequence complementary with miR-142a-3p sequence. [score:3]
Figure S2 Rescue of miR-142a-3p duplex induced hemorrhage phenotype in zebrafish embryos using antisense morpholino targeting to the mature form of miR-142a-3p. [score:3]
Overexpression of miR-142a-3p in zebrafish embryos induces hemorrhage and vascular remo deling. [score:3]
Consistent with these findings, zebrafish with impaired cdh5 function display vascular instability and cranial hemorrhages mimicking those observed in the current study upon suppression of cdh5 by miR-142a-3p. [score:3]
Using an antisense morpholino designed against miR-142a-3p, we successfully rescued miR- 142a-3p overexpression phenotype and demonstrate the specificity of the miRNA -induced phenotype. [score:3]
To investigate the potential target of miR-142a-3p, we focused our analysis on the 672 genes that have putative expression in blood and blood vessel tissue in zebrafish (see previous section on selection of miRNAs). [score:3]
Furthermore we show that the action of miR-142a on cdh5 is in part regulated by Lmo2, an important transcription factor, known for its role in vasculature development. [score:3]
Potential Targets of miR-142a-3p. [score:3]
C - Silencing effect of miR-142a-3p on the GFP-4X PT 3′UTR gene target. [score:3]
Of these, three miRNAs, miR -144, miR-1 and miR-142a-3p revealed specific non-overlapping phenotypes affecting vascular development. [score:2]
In summary, we show that knockdown of lmo2 function in zebrafish embryos leads to an increase in miR-142a-3p transcript levels with a corresponding decrease in the cdh5 transcript and protein levels. [score:2]
In our in silico analysis, we obtained 52 genes that could potentially bind to miR-142a-3p and influence vascular development (Figure 1, Table S4). [score:2]
However in miR-142a-3p knockdown embryos, the intersegmental vessels sprout and extend dorsally but either fails to make contacts with the DLAV (data not shown) or are irregularly patterned (Figure 5D –5G and S1B – S1G). [score:2]
These data demonstrate that morpholino mediated knockdown of endogenous miR-142a-3p in zebrafish embryos do not affect primary vasculogenesis however angiogenesis and intersegmental vessels (Se) remo deling were affected. [score:2]
The negative regulation of miR-142 by the oncogene lmo2 and its co-factors has also been documented in cell culture mo dels [32]. [score:2]
In zebrafish, miR-142a-3p regulates cdh5. [score:2]
Of the eight-selected miRNA tested using zebrafish as a mo del system, we observed specific non-overlapping phenotypes affecting vascular development for three miRNAs, namely miR-1, miR-144 and miR-142a-3p (Figure 2). [score:2]
B–G Representative image of Tg (fli1:EGFP, gata1a: dsRed) zebrafish embryos displaying intersegmental vessels (Se) from non -injected control and 200 µM miR-142a-3p morpholino injected embryos at different developmental stages. [score:2]
0052588.g007 Figure 7In zebrafish, miR-142a-3p regulates cdh5. [score:2]
In zebrafish, miR-142a-3p regulates cdh5 via its two predicted binding sites. [score:2]
Overexpression of miR-142a-3p resulted in the reduction of cdh5 transcript level by approximately 3.4 fold (0.29±0.15) compared to control embryos (Figure 7A). [score:2]
Cdh5 and miR-142a-3p are Regulated by the Transcription Factor Lmo2 in Zebrafish. [score:2]
B - analysis for Cdh5 protein in zebrafish embryos using previously tested antibody directed against human VE-cad (110 kDa) [13] in non -injected control (NIC) and 10 µM miR-142a-3p duplex injected 2 dpf zebrafish embryos. [score:2]
However, to the best of our knowledge distinct functional roles of miR-144, miR-1 and miR-142a-3p in vascular development are yet to be explored. [score:2]
We show that miR-142a-3p could regulate cdh5 gene post-transcriptionally and modulate the levels of cdh5 transcript and protein in zebrafish embryos. [score:2]
Based on our study, we suggest a potential new function of miR-142-3p in regulating the levels of cdh5 required for normal angiogenesis and vascular remo deling in zebrafish embryos. [score:2]
The oncogene lmo2 has been previously shown to transcriptionally regulate both VE-Cadherin and miR-142 in human cell culture mo dels [32], [33]. [score:2]
More recently, the role of miR-142a-3p has been implicated in hematopoiesis, cardiogenesis, and somitegenesis in developing zebrafish embryos through regulating 3′ UTR of rock2a transcript [38]. [score:2]
In Zebrafish, miR-142a-3p Regulates cdh5 In silico miR-142a-3p “seed sequence” search revealed the presence of two potential binding sites in the 3′ UTR of cdh5 gene (Figure 6A). [score:2]
Collectively, this suggests that miR-142a-3p may also be required for development of heart in zebrafish. [score:2]
We were intrigued with the possible biological reasons on why miR-142a-3p should regulate cdh5 gene. [score:2]
miR-142a-3p and Cdh5 are regulated by the transcription factor Lmo2 in zebrafish. [score:2]
In Zebrafish, miR-142a-3p Regulates cdh5. [score:2]
To rule out the possibility of non-specific effect generated by the action of any one of single strand of the miRNA we also conducted separate microinjections of the sense and antisense strand of miR-142-3p. [score:1]
A - Bar graph showing cerebral hemorrhage phenotype in non -injected control (NIC); 10 µM miR-142a-3p duplex injected; co-injection of 10 µM miR-142a-3p duplex with 100 µM miR-142a-3p morpholino (MO); and 100 µM miR-142a-3p MO injected Tg (fli1:EGFP, gata1a: dsRed) zebrafish embryos at 2dpf. [score:1]
This suggests that the 3′UTR of cdh5 specifically interact with miR-142a-3p. [score:1]
A - Sequence alignment of mature miR-142a-3p from different species is highlighted with seed sequence in gray color. [score:1]
0052588.g005 Figure 5A –Relative quantification of miR-142a-3p in GFP positive (+) and GFP negative (–) cells of 2 dpf Tg (fli1:EGFP, gata1a: dsRed) zebrafish embryo by real-time PCR. [score:1]
D,F,H - Non -injected control embryos (NIC) and E,G,I - miR-142a-3p injected embryos. [score:1]
F and G – Embryos receiving co-injection of 10 µM miR-142a-3p duplex with 100 µM miR-142a-3p MO. [score:1]
B – Synteny analysis of human miR-142 on chromosome 17 with zebrafish miR-142a on chromosome 5. Arrow in genes indicates strand information on chromosome. [score:1]
Total RNA was isolated from 2 dpf non -injected control (NIC) and 10 µM miR-142a-3p duplex injected Tg (fli1:EGFP, gata1a: dsRed) zebrafish embryos. [score:1]
Mature miR-142a-3p sequence is in red color. [score:1]
H - Bar graph showing embryos with inter-segmental vessel defects in non -injected control (NIC) and 200 µM miR-142a-3p morpholino injected embryos at 28–30 hpf. [score:1]
Also the miR-142a genomic loci share syntenic relationship between human and zebrafish (Figure 3B). [score:1]
The results of this study reveal an essential role for miR-142a-3p in angiogenesis and vascular remo deling in vivo. [score:1]
Loss of miR-142a-3p leads to intersegmental vessel remo deling. [score:1]
Microinjection of miR-142a-3p MO at concentration of 200 µM caused specific vascular phenotype (Figure S1B – S1G). [score:1]
We also noticed that loss of function of miR-142a-3p resulted in vascular remo deling of intersegmental vessels (Se) in developing zebrafish embryos. [score:1]
MiR-142a-3p has been shown to regulate RAC1 in hepatocellular carcinoma cells [37]. [score:1]
A - Schematic of GFP- vegabf 3′UTR transcript containing two miR-142a-3p predicted binding site (site “A”, blue; site “B”, purple). [score:1]
B – Schematic alignment of miR-142a-3p MO with pre-miR-142a. [score:1]
In silico miR-142a-3p “seed sequence” search revealed the presence of two potential binding sites in the 3′ UTR of vegfab and cdh5 gene (Figure 6A and S3A). [score:1]
A – D - Confocal images (GFP and GFP/RFP merged) of 10 µM miR-142a-3p duplex injected Tg (fli1:EGFP, gata1a: dsRed) embryos at 2 dpf (Dorsal View of head) depicting dorsal midbrain junction (DMJ) and central arteries (CtA) (10× magnification). [score:1]
Microinjection of duplex miR-142a-3p (10 µM) induced cerebral/trunk hemorrhage and pericardial edema in approximately 55% of injected animals (n = 113/206). [score:1]
We observed that 52 genes contain a putative binding site for miR-142a-3p (Table S4). [score:1]
Selected nucleotides were mutated in the seed region for destroying miR-142a-3p binding site is displayed in yellow color. [score:1]
A - Graphical representation of dose dependent microinjection ranging from 10–500 µM of miR-142a-3p morpholino (MO) in 28–30 hpf Tg (fli1:EGFP, gata1a: dsRed) zebrafish embryos. [score:1]
In the middle row of each panel various GFP-4X PT 3′UTR sensor RNA and miR-142a-3p combinations were tested as labeled. [score:1]
Loss of miR-142a-3p Leads to Intersegmental Vessel (Se) Remo deling. [score:1]
N,O,P - Zebrafish embryos injected with miR-142a-3p display pooling of blood cells in head/trunk region. [score:1]
Therefore, we analyzed the effects of blocking the mature form of endogenous miR-142a-3p using a morpholino (MO) antisense oligonucleotide (miR-142a-3p MO). [score:1]
Total RNA was isolated from 3 dpf non -injected control (NIC) and 200 µM miR-142a-3p MO injected Tg (fli1:EGFP, gata1a: dsRed) zebrafish embryos. [score:1]
The western blot analysis of Cdh5 (VE-cadherin) protein showed decrease in endogenous protein level in miR-142a-3p injected zebrafish embryos (Figure 7B) [13] [13]. [score:1]
Zebrafish embryos were coinjected with miR-142a-3p duplex and mRNA encoding GFP fused to the individual miR-142a-3p binding sites in triplicate (GFP-3′UTR 3X site A and GFP-3′UTR 3X site B). [score:1]
C – Co-injection of miR-142a-3p with various test combinations of GFP sensor fused to the miR-142a-3p seed sequence binding sites, engineered in triplicates. [score:1]
B and C - Silencing effect of miR-142a-3p on the series of sensor mRNA encoding GFP fused to various test 3′UTRs (as tabulated in panel D). [score:1]
H and I - 100 µM miR-142a-3p MO injected embryos. [score:1]
A –Relative quantification of miR-142a-3p in GFP positive (+) and GFP negative (–) cells of 2 dpf Tg (fli1:EGFP, gata1a: dsRed) zebrafish embryo by real-time PCR. [score:1]
0052588.g004 Figure 4A – D - Confocal images (GFP and GFP/RFP merged) of 10 µM miR-142a-3p duplex injected Tg (fli1:EGFP, gata1a: dsRed) embryos at 2 dpf (Dorsal View of head) depicting dorsal midbrain junction (DMJ) and central arteries (CtA) (10× magnification). [score:1]
In silico miR-142a-3p “seed sequence” search revealed the presence of two potential binding sites in the 3′ UTR of cdh5 gene (Figure 6A). [score:1]
However upon co-injection of 10 µM miR-142a-3p duplex and 100 µM miR-142a-3p MO, the hemorrhage phenotype was rescued in approximately 50% of embryos (35/129) (Figure S2). [score:1]
0052588.g003 Figure 3A - Sequence alignment of mature miR-142a-3p from different species is highlighted with seed sequence in gray color. [score:1]
We searched for complementary seed sequence binding site of miR-142a-3p in the 3′UTR of 672 genes. [score:1]
C - Relative quantification of mature miR-142a-3p in non -injected control (NIC) and embryos injected with 200 µM miR-142a-3p MOs at 2 dpf. [score:1]
We observed that in embryos receiving 10 µM miR-142a-3p duplex alone, 47% (64/134) of embryos display the hemorrhage phenotype. [score:1]
A - Schematic of GFP- cdh5 3′UTR transcript containing two miR-142a-3p predicted binding site (site “A”, grey; site “B”, brown). [score:1]
Analysis of the organization of vasculature in the head region of miR-142a-3p duplex injected embryos with confocal microscope revealed multiple vascular remo deling defects in the miR-142a-3p duplex injected embryos (Figure 4A–4D). [score:1]
The miR-142a-3p mediated repression on the 3′UTR of cdh5 was abolished when both seed sequences were mutated (GFP-3′UTR 3X mut siteA and GFP-3′UTR 3X mut siteB ) to avoid miRNA seed base pairing (Figure 6C). [score:1]
The miR-142a-3p has been identified in different organisms with 100% similarity at nucleotide sequence level (Figure 3A). [score:1]
The optimal dose of the morpholino was determined by injecting zebrafish embryos with 10–500 µM of miR-142a-3p MO (Figure S1A). [score:1]
Detailed investigation of miR-142a-3p revealed that overexpression of miR-142a-3p resulted in a cerebral/trunk hemorrhage along with abnormal cranial vasculature remo deling in developing zebrafish embryos. [score:1]
Inset displaying intersegmental vessels from non -injected control and 200 µM miR-142a-3p morpholino injected embryos. [score:1]
C - Bar graph showing cerebral hemorrhage phenotype in miR-142a-3p duplex injected Tg (fli1:EGFP, gata1a: dsRed) zebrafish embryos at 2 dpf. [score:1]
Upper panel displays GFP- vegfab 3′UTR sensor RNA and miR-142a-3p combinations. [score:1]
The actions of miR-142a-3p reflect its potential to modulate the permeability and remo deling of the endothelium through interaction with endothelial cell-cell adhesion complex. [score:1]
Figure S1 Microinjection of miR-142a-3p morpholino (MO) in zebrafish embryos in Tg (fli1:EGFP, gata1a: dsRed) leads to intersegmental vessel (Se) remo deling. [score:1]
A, B - non -injected control embryos and C, D - 10 µM miR-142a-3p duplex injected embryos. [score:1]
0052588.g006 Figure 6A - Schematic of GFP- cdh5 3′UTR transcript containing two miR-142a-3p predicted binding site (site “A”, grey; site “B”, brown). [score:1]
We wanted to investigate which of the two predicted binding sites in the 3′UTR of cdh5 gene was targeted by miR-142a-3p. [score:1]
D and E - 10 µM miR-142a-3p duplex injected embryos. [score:1]
The black “X” indicates a mutated miR-142a-3p site. [score:1]
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[+] score: 237
MiR-142a-3p inhibits p53 by direct bindingOn the basis of several studies revealing that p53 could act as a direct target of microRNAs [41] and the increase of p53 in 142 [T−/−] embryos, we sought for the possibility that p53 might act as a direct target of miR-142a-3p. [score:10]
142 [T−/−] embryos display ectopic expression of p53To elucidate whether the expression of p53 in miR-142a mutants is consistent with the microarray data in morphants, we first examined the expression of p53 and its downstream target p21. [score:9]
Overexpression of the miR-142a-3p duplex in HEK293T cells and zebrafish embryos suppressed the luciferase activity of WT but not the mutated p53-coding region in a dose -dependent manner (Figure 6b, Supplementary Figure S6a), indicating that p53 is a direct target of miR-142a-3p. [score:8]
On the basis of several studies revealing that p53 could act as a direct target of microRNAs [41] and the increase of p53 in 142 [T−/−] embryos, we sought for the possibility that p53 might act as a direct target of miR-142a-3p. [score:7]
To elucidate whether the expression of p53 in miR-142a mutants is consistent with the microarray data in morphants, we first examined the expression of p53 and its downstream target p21. [score:7]
On the basis of the decrease in proliferation and the increase in apoptosis of HSPCs in 142 [T−/−] embryos, we referred to our previous microarray data that contained upregulated genes in miR-142a-3p morphants [26] and found that apoptotic genes including p53 were greatly upregulated (Supplementary Figure S5b). [score:7]
Taken together, miR-142a-3p is indispensable for the HSPC emergence and differentiation, and its direct target irf7 is upregulated in 142 [T−/−] embryos, confirming the findings in our previous study [26]. [score:7]
Previous work showed that irf7 is a direct target of miR-142a-3p, and miR-142a-3p morphants display increased expression of irf7 [26]. [score:6]
To further demonstrate the inhibition of p53 by miR-142a-3p directly, we ectopically expressed the full-length p53 cDNA fused with Myc-tag in the HEK293T cells. [score:6]
qPCR showed that knockdown of irf7 or p53 had no effect on the expression of each other (Figure 8c and d), revealing that irf7 and p53 are parallel targets of miR-142a-3p. [score:6]
It was reported that ectopic expression of the miR-142-3p duplex (concentration of 20 μmol l [−1]) suppresses the primitive erythrocyte progenitors (gata1 [+]) and HSC formation (cmyb [+]) [27]. [score:5]
In fact, knockdown of miR-142a-5p by antisense morpholino showed normal expression of HSPC marker runx1 (Supplementary Figure S3c), excluding the involvement of miR-142a-5p in HSPC regulation. [score:5]
In this work, we demonstrate that p53 is a direct target of miR-142a-3p during zebrafish definitive hematopoiesis, and the hematopoietic defects in miR-142a-3p mutants can be rescued by loss of p53, which deepens our understanding of the role of p53 in mediating miR-142a-3p function in HSPC development in zebrafish. [score:5]
In our previous study, suppression of miR-142a-3p by antisense morpholinos shows that HSPC development is impaired when miR-142a-3p is knocked down. [score:5]
In summary, our studies demonstrate that miR-142a-3p directly targets p53 to regulate HSPC survival in zebrafish embryos. [score:5]
In addition to our previous report in which irf7 is a direct target of miR-142a-3p in controlling HSPC formation and differentiation, here we discover that p53 acts downstream of miR-142a-3p to control the survival of HSPCs. [score:4]
To confirm the direct targeting of p53 by miR-142a-3p, we cloned the WT p53-coding region as well as mutated sequences (mismatched with miR-142a-3p seed sequence) containing the predicted miR-142a-3p -binding site into luciferase and GFP reporter vector (Figure 6a, Supplementary Figure S6a and b). [score:4]
Further microarray data and functional assays demonstrate that irf7 acts as a direct target of miR-142a-3p and that the miR-142a-3p-irf7 signal pathway regulates the formation and differentiation of definitive HSPCs possibly through the inflammatory signaling [26]. [score:4]
Consistent with the reporter assays, overexpression of miR-142a-3p could attenuate Myc-P53 expression in WT p53 but not the mutated p53 -transfected cells (Figure 6c and d), further supporting the specific binding of miR-142a-3p to p53. [score:4]
P53 mediates the decrease in HSPCs upon loss of miR-142a-3pTo determine whether p53 upregulation mediated the HSPC defects in 142 [T−/−] embryos, we first injected p53 morpholino (MO) into 142 [T−/−] embryos. [score:4]
In our previous study, irf7 knockdown only partially rescued the HSPC defects in miR-142a-3p morphants [26], suggesting that other targets of miR-142a-3p may be involved in this process. [score:4]
Taken together, these data strongly suggest that p53 acts as a direct target of miR-142a-3p. [score:4]
Studies in Xenopus demonstrated that miR-142a-3p targets TGFβ and functions as a master regulator in HSPC lineage specification and thus sits at the apex of the hierarchy programming definitive hemangioblast [23]. [score:4]
Loss of p53 could rescue the defects of HSPCs in miR-142a-3p mutants and direct binding assay together support that p53 is a direct target of miR-142a-3p, and the newly identified miR-142a-3p-p53 axis has a critical role in HSPC survival and thus protects emerging HSPCs and their derivatives from apoptosis. [score:4]
First, we designed a pair of TALEN arms that target two sites inside of the miR-142a gene (Figure 1a). [score:3]
Decreased HSPCs and their derivatives, as well as ectopic expression of p53 and irf7, were observed in miR-142a-3p mutants. [score:3]
Recently, by targeting two miR-142 genes including miR-142a and miR-142b, the mutants of miR-142 by zinc-finger nucleases (ZFN) method are generated, and these miR-142-3p mutants display neutrophil defects but with relatively normal HSPCs [24]. [score:3]
Further analysis revealed that the expression of miR-142a-3p in both the thymus and caudal hematopoietic tissue region was undetectable (Figure 1e). [score:3]
Consistent with our previous finding that miR-142a-3p is highly expressed in the runx1 [+]/ cmyb [+] cells in the trunk region that labels HSPC populations in zebrafish [26], two independent groups also showed evidence that the counterpart of miR-142a-3p is enriched in HSPC population in Xenopus and mice [22, 23]. [score:3]
In fact, double knockdown of irf7 and p53 could fully rescued the HSPC defects and the rescue effect was better compared with irf7 knockdown, demonstrating that there is a synergistic effect of both irf7 and p53 downstream of miR-142a-3p in HSPC development. [score:3]
MiR-142a-3p inhibits p53 by direct binding. [score:3]
Moreover, injection of GFP reporter fused with the WT p53 but not the mutated p53-coding region together with the miR-142a-3p duplex displayed a marked inhibition of GFP activity (Supplementary Figure S6b). [score:3]
To determine whether there is a synergy effect between irf7 and p53 in miR-142a-3p -mediated HSPC development, we knocked down both irf7 and p53 in 142 [T−/−] embryos. [score:3]
Systematic bioinformatic analysis using RNA hybrid website (BiBiServ2) revealed that there is a miR-142a-3p -binding site in the coding sequence (CDS) but not the 3’-UTR of zebrafish p53, suggesting that miR-142a-3p may be a direct regulator of p53. [score:3]
Moreover, knockdown of irf7 in 142 [T−/−] embryos displayed a partial rescue on HSPCs at both 26 and 36 hpf (Figure 3c), confirming that irf7 functions downstream of miR-142a-3p to mediate HSPC development. [score:3]
Another group has reported that ectopic expression of miR-142-3p leads to the reduction of primitive erythrocyte progenitor cells and HSCs in zebrafish [27]. [score:3]
Double knockdown of irf7 and p53 fully rescued the HSPC defect (Figure 8a and b), demonstrating that irf7 and p53 act synergistically downstream of miR-142a-3p in HSPC development. [score:3]
In addition, the expression of p53 and irf7 was examined in miR-142a mutants injected with irf7 MO or p53 MO, respectively. [score:3]
We also performed miR-142a-3p duplex injection and found that the dose of duplex they used caused nonspecific side effects in embryos, such as heart edema, whereas lower dose (concentration of 5–0 μmol l [−1]) showed increased expression of HSPC marker runx1 at 26 hpf as well as cmyb and rag1 at 5 dpf. [score:3]
Although developmental defects in HSPCs were not examined in miR-142 [−/−] mice, we speculate that a similar HSPC phenotype would be expected at the onset of definitive hematopoiesis. [score:2]
Here, we show that miR-142a-3p directly binds to the coding region of p53 and blocks its function, and then reveals the crucial role of the miR-142a-3p- p53 axis in HSPC survival. [score:2]
As shown in Figure S7b, the defects of differentiated populations including erythrocytes, neutrophils and T cells in 142 [T−/−] embryos were also partially restored by p53 knockdown, supporting that p53 functions downstream of miR-142a-3p in HSPC differentiation. [score:2]
Furthermore, we generated a double knockout of miR-142a-3p and p53 by outcrossing adult 142 [T−/−] with p53 [M214K−/−]. [score:2]
In zebrafish, miR-142-3p modulates neutrophil development by controlling its maturation [24]. [score:2]
These lines of evidence in stem cells give clues that excessive level of p53 might dampen the emergence of stem cells and miR-142a-3p could protect the nascent emerging HSPCs from unwanted apoptosis, and this protection is critical for HSPC development during embryogenesis. [score:2]
Thus, we examined whether miR-142a-3p mutants display such developmental defects. [score:2]
In addition, p53 acts synergistically with the previously reported irf7 downstream of miR-142a-3p in controlling HSPC development [26] (Supplementary Figure S8). [score:2]
Notably, hematoxylin and eosin staining result revealed that the cellular density of neutrophils was decreased during the loss of miR-142. [score:1]
Whole-mount in situ hybridization (WISH) result demonstrated that miR-142-3p was absent in both the thymus and caudal hematopoietic tissue region in 142 [C−/−] embryos (Supplementary Figure S1e), suggesting a loss-of-function of miR-142a-3p. [score:1]
Generation of miR-142a-3p mutants by TALENs and CRISPR/Cas9In order to substantiate our previous findings that loss of miR-142a-3p leads to decreased HSPCs [26], both TALENs and CRISPR/Cas9 were used to generate genetic mutants of miR-142a. [score:1]
According to the sequencing result, 13 base pairs were deleted in the locus of the miR-142a gene (Figure 1b and c) and this disrupted the secondary structure of miR-142a as well as the formation of mature miR-142a-3p (Figure 1d). [score:1]
Impaired secondary RNA structure of miR-142a and failure to form the mature miR-142a-3p also occurred in 142 [C−/−] embryos (Supplementary Figure S1c and d). [score:1]
On the basis of the deficiency of miR -142a and miR-142b genes, there is a possibility that the reported neutrophil hypermaturation in miR-142a/b mutants might be due to the deletion of miR-142b. [score:1]
It was observed that both the cellularity and the volume of the pronephros decreased dramatically in 12-week-old 142 [T−/−] adult fish (Figure 2g), confirming the continuous defect of definitive hematopoiesis in adult zebrafish upon miR-142a deletion. [score:1]
As expected, p53 MO injection led to alleviated apoptosis in both the trunk and AGM region of 142 [T−/−] embryos (Supplementary Figure S7a), strengthening the cause–consequence relationship between p53 -induced apoptosis and HSPC defects in miR-142a-3p mutant. [score:1]
In vitro, mouse fibroblasts were reprogrammed into hemogenic endothelial cells that differentiated into CD45 [+] cKit [+] cells (HSPCs) between day-20 and day-35 culture; in these HSPCs, the highest enrichment of microRNAs including miR-142 was observed [22]. [score:1]
P53 mediates the decrease in HSPCs upon loss of miR-142a-3p. [score:1]
MiR-142 is also an essential regulator of lymphocyte ontogenesis and megakaryopoiesis [20, 25]. [score:1]
In order to substantiate our previous findings that loss of miR-142a-3p leads to decreased HSPCs [26], both TALENs and CRISPR/Cas9 were used to generate genetic mutants of miR-142a. [score:1]
The WT or mutated p53 CDS was co -transfected with the miR-142a-3p duplex. [score:1]
Consistently, real-time quantitative PCR (qPCR) result demonstrated that miR-142a-3p and another member of miR-142a, miR-142a-5p, were severely decreased (Figure 1f), indicating the loss-of-function of miR-142a-3p and miR-142a-5p. [score:1]
The CRISPR/Cas9-generated miR-142a mutants (142 [C−/−]) caused a 986-base-pair deletion, and the structure of mature miR-142a-3p in both mutants was disrupted. [score:1]
Generation of miR-142a mutants and RNA secondary structure prediction. [score:1]
Wild-type or mutated pGL3- p53 CDS co -transfected with negative control or the miR-142-3p duplex (Invitrogen, Shanghai, China) were transfected into HEK293T cells using Lipofectamine2000 (Invitrogen). [score:1]
The injection dose of miR-142a TALEN mRNA was 250 pg per embryo. [score:1]
Therefore, cells in this region including HSPCs underwent excessive apoptosis in both types of miR-142a mutants. [score:1]
Anti-Myc antibody was used as a tag to test the binding affinity of miR-142a-3p to p53. [score:1]
To determine the role of miR-142a-3p in the expansion and differentiation of HSPCs at later stages, was performed and the result showed that cmyb was decreased in the caudal hematopoietic tissue (the equivalent of fetal liver in mammals) from 2 to 3.5 dpf in 142 [T−/−] embryos (Figure 2e). [score:1]
Therefore, loss of miR-142a-3p leads to the decreased HSPCs and their derivatives in both 142 [T−/−] and 142 [C−/−] embryos. [score:1]
Previous studies implicate that loss of miR-142a-3p leads to decreased erythrocytes as well as vascular defects [39, 40]. [score:1]
for miR-142a-3p was conducted at 52 °C and for others were performed at 65 °C with probes including miR-142a-3p, scl, pu. [score:1]
The TALEN-generated miR-142a mutants (142 [T−/−]) had a 13-base-pair deletion and could be verified by sequencing (Supplementary Table S1). [score:1]
Generation of miR-142a-3p mutants by TALENs and CRISPR/Cas9. [score:1]
To confirm the HSPC defects in miR-142a-3p morphants as reported in our previous study [26], the HSPC marker runx1 was examined in 142 [T−/−] embryos and result displayed its reduction in the aorta–gonad–mesonephros (AGM) at 26 and 36 hpf (Figure 2a). [score:1]
Concerning the absence of miR-142a-3p and miR-142a-5p in 142 [T−/−], it is critical to determine whether the decrease in HSPCs is also attributed to the absence of miR-142a-5p. [score:1]
Thus, the role of zebrafish miR-142a-3p in definitive hematopoiesis remains elusive, largely because of different methods used by different groups. [score:1]
Loss of miR-142a-3p leads to decreased HSPCs in zebrafish. [score:1]
In this work, by generating two types of miR-142a-3p mutants using both TALENs and CRISPR/Cas9 methods, we demonstrate that miR-142a-3p has an essential role in definitive hematopoiesis. [score:1]
Therefore, we concluded that the defects of HSPCs in 142 [T−/−] embryos were only attributed to the absence of miR-142a-3p. [score:1]
In Xenopus, miR-142-3p has an essential role in establishing the mesodermal lineage that contributes to both HSC emergence and vasculogenesis [23]. [score:1]
Because of the dual roles of p53 in both cell cycle arrest and apoptosis and the hint from the microarray, we next focused on the relationship between miR-142a-3p and p53. [score:1]
Loss of miR-142a-3p leads to decreased HSPCs in zebrafishPrevious studies implicate that loss of miR-142a-3p leads to decreased erythrocytes as well as vascular defects [39, 40]. [score:1]
To generate a large deletion of the miR-142a gene by CRISPR/Cas9, a pair of gRNAs flanking the miR-142a locus was designed and co -injected into zebrafish WT embryos with Cas9 mRNA (Supplementary Figure S1a). [score:1]
Generation of miR-142a mutants and RNA secondary structure predictionZebrafish miR-142a mutants were generated by TALEN and CRISPR/Cas9, as previously described [54, 55]. [score:1]
Zebrafish miR-142a mutants were generated by TALEN and CRISPR/Cas9, as previously described [54, 55]. [score:1]
After F2 screening, a miR-142a mutant was identified, named 142 [C−/−], and the sequencing result displayed a 986-base-pair deletion in the locus of miR-142a, which was able to be employed to distinguish the WT siblings and mutants by PCR (Supplementary Figure S1b). [score:1]
This result is discrepant with the previously reported neutrophil phenotypes in miR-142a/b mutants [24]. [score:1]
This is consistent with our work and illustrates the crucial function of miR-142a-3p in definitive hematopoiesis across vertebrates. [score:1]
However, there are also different views on the function of miR-142a-3p in HSPCs based on morpholino or duplex injection [27, 40]. [score:1]
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[+] score: 40
In total, 6 up-regulated miRNAs (such as dre-miR-142a-5p, dre-miR-155, dre-miR-29a, dre-miR-457a, dre-miR-223, and dre-miR-338) were joined to the gene regulatory network by potentially targeting relationships with 8 down-regulated genes. [score:10]
Furthermore, the enforced expression of miR-142-3p, a uniquely expressed miRNA, causes a significant decrease in primitive erythrocyte progenitor cells and HSCs. [score:5]
Interestingly, the gata1, cmyb and runx1 expression were all markedly decreased when miR-142-3p was ectopically expressed (Figure 7), indicating that miR-142-3p affects HSC formation in zebrafish. [score:5]
To study the hematopoietic phenotype caused by increased miR-142-3p signaling, we overexpressed miR-142-3p using miR-142-3p duplex overexpression. [score:5]
However, the scl expression was comparable to the control embryos, suggesting that miR-142-3p is not required for early hemangioblast formation. [score:3]
We further confirmed that one of the uniquely expressed miRNAs, miR-142-3p, plays a critical role during erythrocyte progenitor cell and HSC formation. [score:3]
Using O-dianisidine hemoglobin staining, a significant decrease of mature erythroid cells was detected when miR-142-3p was overexpressed. [score:3]
In addition, several hematopoietic miRNA signatures are uniquely expressed in RPS24 MO such as miR-142-3p and miR-29a, which have been previously reported to be required for the formation and differentiation of hematopoietic stem cells [19, 20]. [score:3]
Overall, these data suggest that increased miR-142-3p represses primitive erythroid progenitor cell and HSC formation. [score:1]
miR-142-3p is required for primitive erythroid progenitor cell and HSC formation. [score:1]
Furthermore, all of the above hematopoietic phenotypes were alleviated by co-injection of miR-142-3p mimics and MO (data not shown). [score:1]
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[+] score: 15
Other miRNAs from this paper: dre-mir-196a-1, dre-mir-199-1, dre-mir-199-2, dre-mir-199-3, dre-mir-203a, dre-mir-210, dre-mir-214, dre-mir-219-1, dre-mir-219-2, dre-mir-221, dre-mir-222a, dre-mir-430a-1, dre-mir-430b-1, dre-mir-430c-1, 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-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-21-1, dre-mir-21-2, dre-mir-25, dre-mir-30e-2, dre-mir-101a, dre-mir-103, dre-mir-107a, dre-mir-122, 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-126a, dre-mir-129-2, dre-mir-129-1, dre-mir-130b, dre-mir-130c-1, dre-mir-130c-2, dre-mir-133a-2, dre-mir-133a-1, dre-mir-133b, dre-mir-133c, dre-mir-135c-1, dre-mir-135c-2, dre-mir-140, dre-mir-142b, dre-mir-150, dre-mir-152, dre-mir-462, dre-mir-196a-2, dre-mir-196b, dre-mir-202, dre-mir-203b, dre-mir-219-3, dre-mir-365-1, dre-mir-365-2, dre-mir-365-3, dre-mir-455-1, 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, dre-mir-135b, dre-mir-135a, dre-mir-499, dre-mir-738, dre-mir-429b, dre-mir-1788, dre-mir-196c, dre-mir-107b, dre-mir-455-2, dre-mir-222b, dre-mir-126b, dre-mir-196d, dre-mir-129-3, dre-mir-129-4
Quantification of miRNA and miRNA* expression by qRT-PCR (Figure 5C) confirmed the higher relative abundance of miR-140* and miR-142a* and showed that dre-miR-142* was expressed during development while its mature miRNA was not detected. [score:6]
Dre-miR-142* showed significantly higher expression than its mature miRNA, which was not detected during development. [score:4]
In some ZF samples, the number of miRNA* reads was higher than that of mature miRNA sequences, namely dre-miR-129*, dre-miR-140*, dre-miR-142a*, dre-miR-202*, dre-miR-210* and dre-miR-214*. [score:1]
Dre-miR-142a* had the highest number of sequence reads (391). [score:1]
Similar results were observed before for dre-mir-129*, dre-mir-142a*, dre-mir-142b* and dre-mir-214* [20]. [score:1]
In the case of dre-miR-129, dre-miR-140, dre-miR-142a, dre-miR-202, dre-miR-210 and dre-miR-214, the number of miRNA* reads was considerably higher than that of mature miRNA reads, suggesting that the miRNA* strand was more stable than the miRNA strand. [score:1]
However, six miRNA* were more abundant than their corresponding mature miRNAs, namely dre-miR-129*, dre-miR-140*, dre-miR-142a*, dre-miR-202*, dre-miR-210* and dre-miR-214*. [score:1]
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[+] score: 12
Totally, the final results included 9 downregulated miRNAs (dre-miR-142a-3p, dre-miR-142b-5p, dre-miR-144-3p, dre-miR-146a, dre-miR-190a, dre-miR-219-5p, dre-miR-301b-3p, dre-miR-459-5p and rno-miR-33-5p) and 3 upregulated miRNAs (dre-miR-735-3p, dre-miR-735-5p and mmu-miR-6240). [score:7]
MiR-301 also regulated the expression of key proteins involved in cholesterol homeostasis and blood lipid levels [27], consistent with what was indicated in S3 Fig. Although the research realm of miR-142a centered on cardiovascular and immune system [28, 29], evidences suggested its renal -associated actions [30]. [score:4]
In addition, miR-142a was major constituent of mitochondrion-enriched miRNAs in response to nerve tissue injury [31]. [score:1]
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[+] score: 11
As previously reported, some of the specifically expressed miRNAs in RPL5 MO (such as Dre-mir-142a-3p, Dre-miR-34b and Dre-miR-15a*) and some miRNAs with two-fold higher expression (such as Dre-miR-150, Dre-miR-223 and Dre-miR-155) are involved in the development and function of the hematological system and most of the above-listed miRNAs participate in normal hematologic functions by regulating the expression of c-myb [28– 31]. [score:9]
In addition, many screened miRNAs were hematopoietic-specific, especially dre-miR-142a-3p, which can result in the loss of hematopoiesis during embryonic development in zebrafish [28]. [score:2]
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[+] score: 4
Recently, we reported that expression of miR-142a-3p was restricted to the vasculature endothelium and has a role in developmental angiogenesis in zebrafish [32]. [score:4]
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[+] score: 3
Additionally, several miRNAs are known to regulate cardiogenesis 4– 7. For example, during the early stages of mesoderm formation, miR-142-3p plays a crucial regulatory role in cardiogenesis, hematopoiesis and somitogenesis [6]. [score:3]
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[+] score: 1
Other miRNAs from this paper: dre-mir-142b
Nishiyama T. Kaneda R. Ono T. Tohyama S. Hashimoto H. Endo J. Tsuruta H. Yuasa S. Ieda M. Makino S. miR-142–3p is essential for hematopoiesis and affects cardiac cell fate in zebrafish Biochem. [score:1]
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