Effect Of MiR-29c Overexpression On The Proliferation,Apoptosis And Differentiation Of P19 Embryonal Carcinoma Cells

In vertebrate embryonic development,the heart is the first organ that becomes formational and functional.The intricate process of embryonic development and progression requires precise temporal and spatial regulation of gene expression and multiple signaling pathways,such as Wnt signaling pathway,Notch signaling pathway,BMP signaling pathway,etc.Errors in these processes can result in cardiac development malformations,which produce the most commond forms of congenital heart disease(CHD).Hence,a better understanding of the pathogenesis of the genetic ways,perhaps provide a novel direction for the prevention and treatment of CHD.With the rapid development technology of life science,learning more about the mechanism for the development of many diseases no longer limited to the gene and protein levels.There is a new understanding of the noncoding RNAs.Micro RNAs(miRNAs)are a class of small,endogenous 18–22 nucleotide,stable noncoding RNAs that critically modulate post-transcriptional gene negative regulation by binding to the 3'UTR region of target gene m RNA,resulting in repression of translation or degradation of the target m RNA,reduce expression of the target protein,mediate a variety of biological processes,such as cell growth,proliferation,apoptosis,differentiation,and sequence development,which expand the understanding of miRNAs relate to heart development and heart disease.Numerous studies have confirmed that miRNAs play a key role in various cardiovascular events through their regulation of heart gene expression and function of cardiomyocyte,and their dysregulation is involve in heart disease.Embryonic heart development is tightly regulated by various genes,miRNA is important post-transcriptional factor,but the role of miRNAs in modulating embryonic heart development was still in the initial stage.In the previous study,we used the miRNA array to identify the differential expressed miRNA of aborted fetuses that had ventricular septal defect(VSD),and found an unknown functional miRNA---miR-29 c,highly differential expression in VSD,suggesting that miR-29 c is related to cardiac development malformations.In contrast,few studies have established the function and mechanism of miR-29 c causing embryonic heart malformations.P19 cells and zebrafish have advantages in the use as models for molecular analysis of embryonic heart development.In this context,we mediated the expression of miR-29 c to investigate its effect on P19 cell proliferation,apoptosis,and differentiation,and evaluated its function in zebrafish embryonic heart development and preliminary analyzed the role of miR-29 c downstream targets,to provide a new direction for the role of miR-29 c physiopathologic in embryonic heart development.Part ? Effect of miR-29 coverexpression on the Proliferation,Apoptosis of P19 CellsObjective: To investigate the role of miR-29 c on P19 cell proliferation,apoptosis,and obtain the overexpression of miR-29 c zebrafish embryonic heart development phenotype.Methods: The mmu-miR-29 coverexpression and mmu-miR with GFP control plasmids were transfected into P19 cells and selected by puromycin for two weeks.Stable cells were imaged using fluorescence microscopy and screened using quantitative real-time PCR(q RT-PCR).CCK-8 assay was used to assess the proliferation,flow cytometry was used to analyse the cell cycle and the apoptosis was detected by Hoechst stain,flow cytometry,caspase-3,-8,and-9 activity and BAX,BCL-2 expression level was measured by q RT-PCR and Western blot in miR-29c– overexpressing P19 cells.The miR-29 c minic was microinjected in the zebrafish single-cell stage to observe the morphological change.Results: Overexpressed miR-29 c inhibited proliferation and cell cycles indicated a decrease in the percentage of cells in S-phase.The ratio of BAX/BCL-2 and caspase-3,-8,and-9 activities were significantly increased in miR-29 coverexpressing cells,which indicated increase of the apototsis.The morphological observations showed that overexpression of miR-29 c in zebrafish embryos lead to significant pericardial edema.Conclusion: miR-29 coverexpression affects the proliferation and apoptosis of P19 cells and may lead to pericardial edema in the morphological of zebrafishembryonic heart development.Part 2 Effect of miR-29 coverexpression on the Differentiation and WNT4 signaling pathway of P19 CellsObjective: To explore the effect of miR-29 c on P19 cell differentiation and Wnt4 signaling pathway.Methods: Induce the stably miR-29 cover-expressed P19 cells differentiate into cardiomyocytes.QRT-PCR was used to detect the expression of GATA binding protein 4(GATA4),cardiac troponin T(c Tn T),myocyte enhancer factor 2C(MEF2C)and Wnt4 signaling pathway related genes during differentiation.And related proteins were detected by Western blot.Luciferase assay validated whether Wnt4 is miR-29 c target.Results: Expression of the cardiac-specific markers indicating differentiation increased in miR-29c-overexpressing cells compared to the controls.Luciferase assay confirmed that Wnt4 was a miR-29 c downstream target.Mi R-29 coverexpression possibly affects the P19 cells differentiation by suppressing WNT4/?-catenin signaling.Conclusion: miR-29 coverexpression may affect the differentiation of P19 cells towards cardiomyocytes by the WNT4/?-catenin signaling pathway.