| Coronary artery disease (CAD) is the leading cause of death worldwide, numerous studies have analyzed the involvement of epigenetic mechanism in the development and progression of CAD. However, how and to what extent of the genomic methylation of DNA contributes to the pathogenesis of CAD was still inconclusive.In the first part of our research, the global DNA methylation levels (5-mdC/dG, m6A/A) in peripheral blood leukocytes (PBLs) DNA of 435 individuals (215 cases of CAD and 220 controls) were quantified by chromatography-mass spectrometry, the expression level of 6 genes in peripheral blood were detected via quantitative real-time PCR (qPCR), including three DNA methyltransferases(DNMT1, DNMT3A, DNMT3B) and three lipid metabolism related genes (SREBP-1, HMGCR, MLXIP). In the second section, THP-1 cell line was used as a model of studies in vitro since the formation of foam cells from monocytes is the characteristic in the early atherosclerosis plaque. Human DNMTl full-length cDNA clone were transfected into the THP-1 foam cells, qPCR and Western Blot were used to evatigate the expression alteration of SREBP-1 in vitro. In the third section, in order to evaluated the possible association of candidate Single Nucleotide Polymorphisms (SNPs) in the epigenetic-regulatory gene, DNMTl, with CAD in Chinese Han population. Five tag SNPs (rsl6999593, rs2336691, rs2228611, rs4804494, rs7253062) were analyzed by High Resolution Melt (HRM) method in 476 CAD patients and 478 controls.The primary findings in this research are:1) leukocytes genomic 5-mdC contents were negatively related with age and serum total cholesterol in control Chinese population; 2) The reduction in genomic 5-mdC contents in PBLs was a risk factor for CAD (odds ratio (OR)=2.454,95% confidence interval (CI) 1.665～3.617, adjusted P value=5.789×10-6) while the decrease in m6A content was a protect factor for CAD (OR=0.655,95% CI=0.448～0.957, adjusted P=0.029); 3) The mRNA expression level of DNMT1 was elevated while SREBP-1 was decreased in CAD patients than that of the controls; 4) Genomic 5-mdC contents were decreased remarkably in THP-1 foam cells, the mRNA expression level of DNMT1 was reduced and SREBP-1 was increased during the foaming process in THP-1 cell line; 5) Plasmid with human DNMT1 full-length cDNA clone was transfected into THP-1 macrophage, the methylation level of SREBP-1 promoter was elevated, while both mRNA expression level and protein expression level of SREBP-1 were decreased; 6) There were significant differences in the genotype and allele distributions of rs2228611 and rs2336691 in DNMT1 between CAD patients and healthy controls; 7) rs7253062 was associated with blood lipids regulation; 8) Haplotypes G-T-A-A-T (alleles in order of rs2228611, rs16999593, rs7253062, rs2336691 and rs4804494) was associated with increased risk of developing CAD.In summary, aberrant DNA methylation was implicated in CAD. The SNPs in DNMT1 and contents of 5-mdC/dG and m6A/A were highly associated with the risk of CAD. Both genome-wide hypomethylation and CpG island hypermethylation were observed during the foam cells formation in THP-1 cells supported that DNA hypomethylation in PBLs could be a contributor to the pathogenesis of CAD. |
