The Discussion Of Methylation Modification In Induced Stem Cell Differentiation To Cardiomyocytes

Objectivethe Discussion of methylation modification in induced Stem cell differentiation to cardiomyocytesMethod1. The experiment was divided into 3 groups: the blank group( just C3H10T1/2), the Lv-GFP group(Control group), and the Lv-islet-1 group(cells infected with a plasmid overexpressing Islet-1/GFP). The Lv-islet-1 group was further divided into the subgroups Islet-1-1 week, Islet-1-2 weeks, Islet-1-3 weeks, and Islet-1-4 weeks based on the lentiviral infection time.2. Fluorescence microscopy was used to observe the GFP expression and morphological changes. The infection efficiency was assessed with flow cytometry. Expression of c Tn T detected by immunofluorescence. Real-time PCR detect the the expression of cardiomyocyte-specific early-stage transcription factors : Nkx2.5, GATA4, and Mef2 c. Using Chromatin immunoprecipitation to detecte histone acetylation sequential changes of GATA4 and Nkx2.5 promoter region. Using the Methylation-specific PCR to detect DNA methylation sequential changes of this cardiomyocyte-specific early-stage transcription factors promoter region.Result1.The GFP fluorescence results indicated that the lentiviral infection was successful. The flow cytometry results showed that the infection efficiency reached 91.7%. The results ensured the reliability of subsequent experiments. The Western blotting results indicated that the C3H10 T1/2 cells had a high level of Islet-1 expression after lentiviral infection compared to the blank group and the control group. After Islet-1 infection, the MSCs exhibited a short rod-shaped morphology and had a homogenous direction, a tight arrangement, a strong refraction. The immunofluorescence results indicated that the MSCs expressed the cardiomyocyte-specific protein c Tn T in the cytoplasm after Islet-1 infection 4 weeks. The detection of cardiomyocyte-specific early-stage transcription factors indicated that the expression of Nkx2.5, GATA4, and Mef2 c gradually increased with the progression of the infection time,and the highest in the Islet-1-3W(*: P < 0.05).2. The MS-PCR results showed that the methylation level of the Cp G sites on the GATA4 promoter(1329 bp-1489 bp) gradually decreased after Islet-1 infection,the decrease was significant at week 3(*: P < 0.05).The methylation levels of the Cp G sites on the Nkx2.5 gene promoter were higher but did not exhibit differences compared to the blank group and Lv-GFP group. The Ch IP-q PCR results showed that the histone methylation levels on the GATA4 and Nkx2.5 gene promoter regions. The bingding of Gata4 and Nkx2.5 combined with H3K9me2 in the C3H10T1/2 cells infected with Lv Islet-1 gradually decreased, and significant at week 3(*: P < 0.05).3. The Ch IP-q PCR results showed that the histone methylation levels on the GATA4 gene promoter regions. The bingding of Gata4 combined with H3K9me2 in the Islet-1 group infected with 5-Aza were significantly lower than those in the Lv Islet-1 group(*: P<0.05). The MS-PCR results showed that the methylation level of the Cp G sites on the GATA4 promoter(1329 bp-1489 bp) in the Islet-1 group infected with BIX01294 were lower than those in the Lv Islet-1 group(*: P<0.05).Conclusion1. The results the DNA methylation level of Cp G sites on the GATA4 promoter during the differentiation of MSCs into cardiomyocyte-like cells inducd by Islet-1 was negatively correlated with the GATA4 expression level. Nkx2.5 expression might not be affected by DNA methylation.2.The histone H3K9me2 level on the GATA4、Nkx2.5 promoter was negatively correlated with the m RNA level.3. the GATA4 histone H3K9me2 levels had positive correlations with GATA4 DNA methylation levels.