Expression Profile And Functional Study Of Micro RNAs During The Differentiation Of Embryonic Stem Cells Into Cardiomyocytes

Aims: Embryonic stem cells(ESCs),derived from the inner cell mass of the blastocyst,are self-renewing cells with the ability to differentiate into diverse cell types of all three germ layers,including functional cardiomyocytes.ESCs hold great potential for the regenerative treatment in cardiac disease.Elucidation of the mechanism underlying the myocardial differentiation from ESCs is essential for therapeutic applications.micro RNAs(miRNAs)are small non-coding RNAs that act as posttranscriptional regulators of gene expression.Emerging evidence has shown that miRNAs play a critical role in the regulation of cardiomyocyte differentiation.However,the specific miRNAs that control ESC differentiation are still largely unexplored.In this study,we aimed to investigate the function of miRNAs in the regulation in the differentiation of ESCs into cardiomyocytes and the underlying mechanism.Methods: We induced mouse ESCs(m ESCs)into cardiomyocytes by embryoid body model,and then detected miRNA expression patterns in mesodermal cells,cardiovascular progenitors,and cardiomyocytes at early differentiation stage and intermediate differentiation stage using miRNA microarray assay.miRNA expression patterns were then validated by realtime-quantitative PCR(Q-PCR).Gain-of-function and loss-of-function studies were performed by establishing miR-142-3p over expression ES cell lines and transiently transfection of miR-142-3p inhibitors respectively.Detection of alkaline phosphatase activity pluripotency gene expression was carried out to examine the function of miR-142-3p on ESC self-renewal.MTT assay,Brd U incorporation,and cell cycle analysis were used to examine the function of miR-142-3p on ESC proliferation.The expression of cardiac specific and germ layer specific genes were evaluated to examine the function of miR-142-3p on cardiac differentiation and germ layer specification.In silico analysis and luciferase reporter assay were carried out to identify direct target genes of miR-142-3p.Results: We showed the miRNA expression patterns during the process of mesoderm,cardiac progenitors,and cardiomyocyte differentiation.Q-PCR validated that the expression of miR-672 in mesodermal cells,miR-142-3p in mesodermal cells and cardiovascular progenitor cells,and miR-193,miR-196 a,miR-196 b,miR-467 e in cardiomyocytes was downregulated.One of these miRNAs,miR-142-3p,was found down-regulated in both mesodermal and cardiac progenitor cells in comparison with their counterparts.The expression of miR-142-3p decreased upon differentiation and then increased from differentiation day 4.Knockdown of miR-142-3p did not affect m ESC self-renewal and cardiac differentiation.Overexpression of miR-142-3p did not affect m ESC self-renewal and proliferation;however,it remarkably repressed cardiomyocyte differentiation.Furthermore,overexpression of miR-142-3p did not affect the expression of germ layer specific genes;however,it decreased the expression of the cardiac mesodermal marker gene Mesp1 and the downstream cardiac transcription fectors Nkx2.5,Tbx5,and Mef2 c.In addition,miR-142-3p neither directly binds to the 3'UTR of Mesp1 nor decreases the known upstream Brachyury,Eomes,and WNT signaling pathway.Finally,Acvr2 a,Rarg,Tgfbr2,Tpm4,Atp2a2,Tgfb2,and Mef2 c,which are closely related with cardiac differentiation or cardiomyocyte contractility,were identified as the candidate targets of miR-142-3p.Conclusion: We revealed the miRNA expression patterns during differentiation of m ESCs into cardiomyocytes.Several miRNAs were found to be involved in the regulation of cardiac differention.miR-142-3p does not affect m ESC self-renewal and proliferation,however,it represses differentiation of m ESCs into cardiomytes likely via inhibiting the expression of Mesp1 during the differentiation process.Regulation of Mesp1 by miR-142-3p is independent of both the known genes Brachyury,Eomes,and WNT signaling pathway.miR-142-3p may function by directly targeting a group of genes that are closely related with cardiac differentiation or cardiomyocyte contractility.These findings may provide new clues for further understanding of the mechanisms and roles of miRNAs as key regulator of cardiomyocyte differentiation.