Study On DNA Methylation Alteration Of Vascular Smooth Muscle Cell Proliferation Related Genes In Type2Diabetic Macrovascular Complications

Objective-Type2Diabetes (T2D) is the majority of all types of diabetes. Its macrovascular complications like cardiovascular diseases and stroke etc., characteristic by atherosclerosis, are the primary cause of high disability rate and significantly increased fatality rate. But the underlying mechanism has not been clearly explained yet. In this study, statistic methods like ROI analysis, GO and Pathway analysis were used to investigate the former raw result of DNA methylation microarray, which was applied in T2D patients accompanied with macrovascular complications. These findings of DNA methylation alteration in specific gene loci and its associated signaling pathways will be meaningful in seeking early methylation signatures to predict diabetic macrovascular complications.Methods-Peripheral blood samples were collected from3groups of T2D patients:patients without complications (n=3), patients with diabetic coronary heart disease (n=3) and patients with diabetic stroke (n=3). DNA methylation was measured by microarrays using methylated DNA immunoprecipitation (MeDIP) method, thus result in raw data and peak values of gene promoters and CpG sites. ROI analysis is a method using median log2-ratio of the individual probes covering defined regions to evaluate enrichment differences, and got the result of statistically different CpG sites in specific gene promoters(p<0.05). Those corresponding genes then went through GO and pathway analysis using Fisher’s exact test to find statistical difference in protein biologicalprocess(BP), cellular component(CC),molecular function(MF) and signaling pathway. David bioinformatics resources6.7and KEGG pathway database were applied to identify the correlation between the genes and signaling pathway in diabetic macrovascular complications. Results-Weidentified139CpG sites in gene promoters with differential DNA methylation between T2D patients without complication and those with coronary artery diseases,381CpG sites compared to diabetic stroke patients. There are59CpG sites had significantly statistical difference in both comparisons. Differential genes enrich the most in the T2D-coresponding pathways including focal adhesion pathway, MAPK signaling pathway and insulin signaling pathway.Conclusion-Our study suggests59differential DNA methylated CpG sites in specific gene promoter, most likely associated with T2D macrovascular complications by MeDIP-DNA methylation microarray. Pathway analysis shows these identified genes participate in several signaling pathway closely related to diabetic complication. Objective-Vascular smooth muscle cells(VSMC) are an important cell component participating in the early genesis of atherosclerotic plaques. Here, we analyzed DNA methylation changes in the promoter CpG islands of VSMC proliferation related genes AKT1and ELK1, and its corresponding alteration in mRNA and protein expression.Methods-The peripheral blood samples were collected from3groups of T2D patients: patients without complications (n=8), patients with diabetic coronary heart disease (n=8) and patients with diabetic stroke (n=8). And blood samples from another8healthy nondiabetic adults were used as control group. Methylation-specific PCR was applied to identify the methylation status of promoter CpG islands in AKT1and ELK1gene. We further investigate AKT1and ELK1gene mRNA expression in all32specimens by means of quantitative real-time PCR method, and enzyme-linked immunosorbent assay(Elisa) was used to detect serum Aktl and Elk-1protein concentration.Results1. The promoter CpG island of gene AKT1exhibited an decreased DNA methylation status in specimens from T2D patients with marcrovascular complications compared with those patients without complications. AKT1mRNA expression was significantly increased in specimens from T2D patients with marcrovascular complications compared with those patients without complications (p<0.001) and correlated positively with serum Aktl protein concentration(p<0.01).2. The promoter CpG island of gene ELK1showed an decreased DNA methylation status in specimens from T2D patients with marcrovascular complications compared with those patients without complications. ELK1mRNA expression was significantly increased in specimens from T2D patients with marcrovascular complications compared with those patients without complications (p<0.001). However, there was no significant differences in serum Elk-1protein level between all groups.Conclusion-Epigenetic modifications like DNA methylation alteration may play a role in the development of macrovascular complications in type2diabetes, given that gene AKT1and ELK1promoter CpG islands exhibited demethylation in T2D patients with coronary artery disease and/or cerebrovascular disease. Overall, detection of gene DNA methylation change may contribute to early clinical diagnosis of T2D macrovascular complications.