Research On Epigenetic Regulation Of Early Myocardial Developmental Gene Expression During Induced Stem Cells Differentiation

ObjectiveDuring Islet-1induced stem cell C3H10T1/2 to differentiate specifically into cardiomyocytes,which is infected by Islet-1 lentivirus vector,through detecte early myocardial developmental gene histone acetylation sequential changes of promoter region and DNA methylation sequential changes of promoter region,to elucidate the interaction between the both.Methods1.Plate the C3H10T1/2 cells in 15 cm2 flasksa at the cell density of 5×104/bottle.when the cell proliferation converged to 60%,use the corresponding amount of Islet-1 expression lentivirus to infected cells in Moi = 20.Detect cell transfection efficiency,expression of GFP fluorescent protein,Islet-1 protein and cardiac-specific gene,and observe the morphological changes.use chromatin immunoprecipitation(CHIP) to detecte histone acetylation changes of early myocardial developmental gene promoter region and the methylation-specific PCR(MS-PCR)to detect DNA methylation changes of early myocardial developmental gene promoter region.2.To treat cells with 5-aza,which was infected with expression Islet-1 lentivirus, use CHIP to detecte histone acetylation changes of early myocardial developmental gene promoter region;To treat cells with TSA,which was infected with expression Islet-1 lentivirus,use the MS-PCR to detect DNA methylation changes of early myocardial developmental gene promoter region.Results1.C3H10T1/2 infected high expression of Islet-1 lentivirus can be detected by fluorescent protein(GFP) and Islet-1 protein expression in cells, transfection efficiency was 95.4%.2.After 3 weeks of induction,closely arranged and trend to substantially coincides fusiform cell population can be observed at a plurality of field of vision.Sequential cardiac cell-specific gene expression in cells after induction.3.With the expression of the GATA4 gene increased,the level of histone acetylation raised,while the DNA methylation level decreased in its promoter.4.The level of histone acetylation increased of GATA4 promoter raised by 5-aza treated cells;The DNA methylation level decreased in the second Cp G site of GATA4 promoter by TSA treated cells.ConclusionsDuring the Islet-1 promotes cells into cardiac-specific differentiation process,it is indicated that histone acetylation and DNA methylation exist mutual antagonism in regulation of the process of early myocardial specific gene expression,which promoted its sequential expression.