Study On The Hydroxymethylation Level Of CCNA2 And TBC1D1 Gene Promoters In Peripheral Blood Of Patients With Early-onset Coronary Heart Disease With Blood Stasis Syndrome

In recent years,the morbidity and mortality of coronary heart disease are increasing rapidly and showing a trend of younger age.Coronary heart disease is caused by genetic factors and environmental factors interact complexity disease,DNA methylation and hydroxyl methylation is one of the important epigenetic modification form,DNA methylation is related to coronary heart disease,and between DNA methylation and hydroxyl methylation and mutual transformation,we hypothesized that hydroxyl DNA methylation modification may participate the premature coronary heart disease(PCHD)related gene expression.Objective:To investigate the genomic DNA hydroxymethylation pattern in peripheral blood from patients with PCHD of blood stasis syndrome(bss)and normal individuals,Screening of hydroxymethylated differentially expressed genes.To explore the DNA hydroxymethylation status in the promoter of CCNA2 and TBC1D1 and validate the result of hMeDIP-chip for uncovering the role of DNA hydroxymethylation in the pathogenesis of bss of PCHD.Methods:We collected six samples each group.bss of PCHD group,PCHD non-bss group,non-PCHD bss group and normal group.hydroxymethylation immunocoprecipitation was used to detect DNA hydroxymethylation levels in promoter regions of CCNA2 and TBC1D1 genes.Result:Through hMeDIP-chip,Compared with the normal group,there were 1070 deps in the group with bss of PCHD group and 702 deps occurred in HCP.GO analysis showed that differences in DNA hydroxymethylation in bss of PHCD changed significantly,the genes were mostly related to gene transcription regulation,cell metabolism and proliferation and other contents.KEGG pathway analysis showed that,DNA methylol-differentially expressed genes in bss of PCHD syndrome were mostly involved in cell metabolismandembryonicdevelopment.CCNA2(PeakScore=2.28,PeakDMValue=0.2598529),TBC1D1gene(PeakScore=2.79,PeakDMValue=0.3523533)and P values of the pathways were significant,in the AMPK signaling pathway enrichment diagram.results of hMeDIP-qPCR showed that,compared with the normal group,CCNA2 gene promoter DNA was hypermethylated(%experimental group/control group=2.286,p=0.0038),while TBC1D1 gene promoter DNA was no different from that of normal people(%experimental group/control group=0.808,p=0.4328).Conclusion:DNA hypermethylation in the promoter region of peripheral blood of PCHD with bss may be related to AMPK signaling pathway,and hypermethylation may be one of the mechanisms leading to the occurrence and development of PCHD with bss.PCR confirmed that there was no significant difference in hydroxymethylation level in the promoter region of TBC1D1 gene,while the high hydroxymethylation of CCNA2 gene promoter DNA was consistent with the results of hMeDIP-chip.