miR-142-3p is essential for hematopoiesis and affects cardiac cell fate in zebrafish.
ABSTRACT MicroRNAs (miRNAs) play a pivotal role during embryonic development and are required for proper organogenesis, including hematopoiesis. Recent studies suggest that, in the early mesoderm, there is an interaction between the hematopoietic and cardiac lineages. However, whether miRNAs can affect other lineages remains unknown. Therefore, we investigated whether hematopoietic miR-142-3p modulated the mesoderm formation. We report that knockdown (KD) of miR-142-3p, a hematopoietic-specific miRNA, in zebrafish resulted in loss of hematopoiesis during embryonic development. Intriguingly, we observed abnormal cardiac phenotypes and insufficiency of somitegenesis in KD-morphants. In the early developmental stage, a tiny heart, contractile dysfunction in the ventricle, cardiac arrhythmia (e.g. a 2:1 ratio of atrial:ventricular beating), and bradycardia were consistently observed. Histological examination revealed severe hypoplasia of the ventricle and disrupted muscle alignment. To determine the mechanism, we performed DNA microarray analysis. The results revealed that the expression of several mesodermal genes essential for the formation of cardiac and somatic mesoderm, such as no tail, T-box gene 16, mesoderm posterior a, one eye pinhead, and rho-associated, coiled-coil containing protein kinase (Rock2a), were increased in miR-142-3p KD-morphants. The luciferase reporter assay revealed that miR-142-3p repressed luciferase activity on the Rock2a 3'-UTR. The findings of the present study indicate that miR-142-3p plays a critical role in hematopoiesis, cardiogenesis, and somitegenesis in the early stage of mesoderm formation via regulation of Rock2a.
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ABSTRACT: MicroRNAs are differentially expressed in cells and regulate multiple biological processes. We have been analyzing comprehensive expression patterns of microRNA in human and mouse embryonic stem and induced pluripotent stem cells. We determined microRNAs specifically expressed in these pluripotent stem cells, and miR-142-3p is one of such microRNAs. miR-142-3p is expressed at higher levels in induced pluripotent stem cells relative to fibroblasts in mice. Level of expression of miR142-3p decreased during embryoid body formation from induced pluripotent stem cells. Loss-of-function analyses of miR-142-3p suggested that miR-142-3p plays roles in the proliferation and differentiation of induced pluripotent stem cells. CpG motifs were found in the 5' genomic region of the miR-142-3p; they were highly methylated in fibroblasts, but not in undifferentiated induced pluripotent stem cells. Treating fibroblasts with 5-aza-2'-deoxycytidine increased the expression of miR-142-3p significantly and reduced methylation at the CpG sites, suggesting that the expression of miR-142-3p is suppressed by DNA methylation in fibroblasts. Luciferase analysis using various lengths of the 5' genomic region of miR142-3p indicated that CpGs in the proximal enhancer region may play roles in suppressing the expression of miR-142-3p in fibroblasts.Stem cell International 12/2014; 2014:101349. DOI:10.1155/2014/101349 · 2.81 Impact Factor
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ABSTRACT: MicroRNAs are endogenous, small non-coding RNAs approximately 18-26 nucleotides in length that regulate target gene expression at the post-transcription level. Interferon-γ (IFN-γ) is a Th1 cytokine that is involved in both the innate and adaptive immune responses. We previously identified two IFN-γ genes in green-spotted puffer fish (Tetraodon nigroviridis). To determine whether miRNAs participate in IFN-γ-related immune responses, T. nigroviridis spleen cells were treated with recombinant IFN-γ isoforms, and a Solexa high-throughput sequencing method was used to identify miRNAs. In total, 1,556, 1,538 and 1,573 miRNAs were found in the three samples, and differentially expressed miRNAs were determined. In total, 398 miRNAs were differentially expressed after rIFN-γ1 treatment, and 438 miRNAs were differentially expressed after rIFN-γ2 treatment; additionally, 403 miRNAs were differentially expressed between the treatment groups. Ten differentially expressed miRNAs were chosen for validation using qRT-PCR. Target genes for the differentially expressed miRNAs were predicted, and GO and KEGG analyses were performed. This study provides basic knowledge regarding fish IFN-γ-induced miRNAs and offers clues for further studies into the mechanisms underlying fish IFN-γ-mediated immune responses.PLoS ONE 05/2014; 9(5):e96336. DOI:10.1371/journal.pone.0096336 · 3.53 Impact Factor
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ABSTRACT: Multiple genetic studies have implicated the autophagy-related gene, ATG16L1, in the pathogenesis of Crohn disease (CD). While CD-related research on ATG16L1 has focused on the functional significance of ATG16L1 genetic variations, the mechanisms underlying the regulation of ATG16L1 expression are unclear. Our laboratory has described that microRNAs (miRNAs), key regulators of gene expression, are dysregulated in CD. Here, we report miRNA-mediated regulation of ATG16L1 in colonic epithelial cells as well as Jurkat T cells. Dual luciferase reporter assays following the transfection of vectors containing the ATG16L1 3'-untranslated region (3'UTR) or truncated 3'UTR fragments suggest that the first half of ATG16L1 3'UTR in the 5' end is more functional for miRNA targeting. Of 5 tested miRNAs with putative binding sites within the region, MIR142-3p, upon transient overexpression in the cells, resulted in decreased ATG16L1 mRNA and protein levels. Further observation demonstrated that the luciferase reporter vector with a mutant MIR142-3p binding sequence in the 3'UTR was unresponsive to the inhibitory effect of MIR142-3p, suggesting ATG16L1 is a gene target of MIR142-3p. Moreover, the regulation of ATG16L1 expression by a MIR142-3p mimic blunted starvation- and L18-MDP-induced autophagic activity in HCT116 cells. Additionally, we found that a MIR142-3p inhibitor enhanced starvation-induced autophagy in Jurkat T cells. Our study reveals MIR142-3p as a new autophagy-regulating small molecule by targeting ATG16L1, implying a role of this miRNA in intestinal inflammation and CD.Autophagy 01/2014; 10(3). DOI:10.4161/auto.27553 · 11.42 Impact Factor