Effects And Mechanisms Of The Upregulation Of MiR-222 And MiR-424/424* In The Development Of Heart

MicroRNAs (miRNAs) are a kind of approximately 22-nuleotide-long noncoding RNAs. During miRNA maturity, Only one strand is usually incorporated into the RNA-induced silencing complex (RISC). Sometimes, either strand of the duplex may potentially act as a functional miRNA, one of them is called miRNA, the other is called miRNA-star. Mature miRNAs regulate target genes by inhibiting translation through imperfect base-pairing or degrading their target mRNAs through near-perfect base pairing.Heart is the first functional organ in vertebrate embryos, every stage of heart development is under control of important genes and signaling paths. Studies in cells or animal models have shown that miRNAs play important roles in cardiomyocyte proliferation, differentiation, and maturation of the heart. Increasing evidences have also revealed that miRNAs are implicated in the congenital heart defects (CHDs) and adult cardiovascular diseases, such as cardiac hypertrophy and heart failure.During previous research, Affymetrix GeneChip miRNA Array was used to detect miRNA expression of TOF and normal heart tissues. Further analysis was detected by qRT-PCR and confirmed that 18 miRNAs were expressed at significantly different levels including miR-222 and miR-424/424*. We also have identified that NF1 and HAS2 was the target gene of miR-424/424*.Part one Effects and Mechanisms of the upregulation of miR-222 in the Development of HeartObjectionThis research is to study the effects and mechanisms of miR-222 on heart development.MethodsPre-miR-222s were injected into zebrafish embryos to upregulate the expression of miR-222. Anatomical microscope and H&E stained heart sections were used to observe the cardiac phenotypes of zebrafishes. Primary embryonic cardiomyocytes were isolated and cultured to detect the effects of miR-222 on cell proliferation by CCK-8 and cell migration by scratch assay. P19 cells were induced by hanging drop method to detect the effects of miR-222 on cardiogenic differention. Bioinformatic analysis predicted the target genes of miR-222. Using luciferase assay, qRT-PCR and western blot, we analyzed the expression of target genes by upregulation of exogenous miR-222 and downregulation of endogenous miR-222. Using qRT-PCR, we analyzed whether Zfpm2 could regulate the expression of Gata4 by upregulation and downregulation of Zfpm2 mRNA in H9C2 cells. QRT-PCR was used to detect the expression of target genes of miR-222 in zebrafishes.ResultsThe miR-222-upregulation zebrafishes owed decreased contractility,the hearts of which are pulled into a string-like structure. Some of the miR-222-upregulation embryos suffer severe edema and noticeable dilation of the atria and/or ventricles. miR-222 promoted cell proliferation in primary embryonic cardiomyocytes and inhibited cardiomyogenic differentiation of P19 cells. ZFPM2(Zfpm2)was the target gene of miR-222 by computational prediction and it was identified by luciferase assay, qRT-PCR and western blot. The upregulation of Zfpm2 promoted the expression of Gata4 in H9C2. The expression of Zfpm2a and Gata4 in miR-222-upregulation zebrafishes was downregulated by miR-222.ConclusionmiR-222 indirectly inhibited Gata4 expression via directly targeting Zfpm2. Upregulation of miR-222 leads to abnormal heart development by targeting the Zfpm2-Gata4 axisPart two The construction of MIR424 transgenic mouse and the effects of miR-424/424* on heart developmentObjectionThis research is to generate MIR424 transgenic mouse and to study the effects of miR-424/424* on heart development.MethodsIZEG-MIR424 plasmid was constructed and tested by X-Gal staining, EGFP observing and qRT-PCR. MIR424 transgenic mouse were got by fertilized eggs injection of the male pronucleus. PCR of genomic DNA and tissue X-Gal staining were used to identify the MIR424 transgenic mouse. We detected the upregulation efficiency of miR-424/424* in MIR424/Nkx2.5 transgenic mouse with qRT-PCR. The heart morphology of MIR424/Nkx2.5 transgenic mouse were observed by HE staining. Primary embryonic cardiomyocytes were isolated and cultured to detect the effects of miR-424/424 on cell proliferation by CCK-8 and cell migration by scratch assay. P19 cells induced by hanging drop method to detect the effects of miR-424/424 on cardiogenic differention.ResultsWe constructed the IZEG-MIR424 plasmid successfully:it could express miR-424/424* in present of Cre enzyme. Only one mouse was identified to be the MIR424transgenic mouse by PCR of genomic DNA and tissue X-Gal staining. The expression of miR-424/424* was upregulated 0.5 fold in MIR424/Nkx2.5 transgenic mouse. And the HE staining did not show any cardiac abnormalities of MIR424/Nkx2.5 transgenic mouse. miR-424/424 promoted cell proliferation and inhibited cell migration in primary embryonic cardiomyocytes. miR-424/424* had no effect on differentiation of P19 cells.ConclusionAlthough MIR424 transgenic mouse were constructed successfully, further analysis of MIR424/Nkx2.5 transgenic mouse is need.