Regulation Mechanism Of SNPs And DNA Methylation On The Expression Of ABCA1 In Promoter Region Of Patients With Ischemic Stroke

Background:Ischemic stroke(IS)is a clinical disease caused by obstruction or stenosis of the supplying arteries.The phenotype of the disease is ischemia,hypoxia or necrosis in the perfusion area.China belongs to the high incidence country of IS.And its high morbidity,high disability and high mortality bring heavy burden to patients,patients’ families and society.The formation and development of atherosclerosis(AS)is the important pathophysiological mechanism of IS.And the key step is the circulating monocytes accumulated at the injured intima and then adhered to the vascular endothelium.Foam cells is a typical pathological cell in early atherosclerosis.Reverse cholesterol transport(RCT)plays an important role in maintaining intracellular lipid balance and preventing the formation of AS.ABCA1 is a key protein in the formation of mature high density lipoprotein and it can inhibit the inflammatory response during atherosclerosis.DNA methylation provides a new way for us to study the pathogenesis of diseases.It participates in the development of diseases by affecting gene expression.DNA methylation is a process by which methyl groups are added to the cytosine in DNA molecule,forming the 5-methylcytosine.Methylation usually occurs on cytosine in Cp G islands.And the promoter region is usually not methylated.However,the level of the methylation in promoter region will change in some disease.It can regulate the gene expression.Gomez-Uriz et al reported that the methylation levels of 80 Cp G loci were found to be different in stroke patients by methylation microarray.We have found that the methylation level of the promoter region of ABCA1 in case group was significantly lower than that in control group.We inferred that the methylation level in the promoter region of ABCA1 may regulate ABCA1 expression.It affects the clearance rate of lipid and cholesterol in cells,and then participates in the pathological process of IS.Studies have shown that single nucleotide polymorphisms(SNP)may affect DNA methylation level in genomic Cp G islands or regulate gene expression.And then it can participate in the development of diseases.However,the effects of SNP loci of ABCA1 on the methylation level of Cp G and the gene expression in ischemic stroke patients remains to be further explored.In conclusion,we hypothesized that SNPs in the promoter region of ABCA1 gene affect the DNA methylation level of Cp G loci of ABCA1 gene.It regulates ABCA1 gene expression and participates in the pathological process of IS by influencing the clearance rate of lipid and cholesterol in cells.This study will reveal the effects of SNPs and DNA methylation on ABCA1 gene expression,providing a new theoretical basis for the genetic mechanism and prevention of the disease.Materials and Method:Part1: SNPs and DNA methylation in promoter region of ABCA1 gene regulate the expression of peripheral blood RNA in ischemic stroke patientsStudy Population: The study protocol was authorized by the Ethics Committee on Human Research of Zhengzhou University,the First Affiliated Hospital of Henan University of Chinese Medicine.And all the subjects were Henan Han population and signed the informed consent.1.The study population for examining the mRNA expression level of ABCA1.The case group selected 100 patients with IS(54 males and 46 females)in the First Affiliated Hospital of Henan University of Chinese Medicine from October 2017 to March 2018.The diagnostic results of all patients were based on the diagnostic criteria of IS established by WHO and confirmed by various examinations(medical history,physical signs,biochemical tests,CT or MRI etc.).The patients with valvular heart disease,atrial fibrillation,hematological diseases,malignant tumors,epilepsy and bacterial infections were excluded.The control group selected 100 individuals(48 males and 52 females)who had physical examination with IS patients in the same time.2.The study population for methylation and SNP genotyping in promoter region of ABCA1 gene.This part of the study population is the same as the population of ABCA1 gene expression.Methods: 1.Total RNA in peripheral blood was extracted by No.DP443 high-efficiency blood total RNA extraction kit of Beijing Tiangen Company.All processes according to the manufacturer’s instructions.Total RNA(300ng for each participant)was reversely transcribed into complementary DNA(c DNA)by Fast King RT Kit of Beijing Tiangen Company.The expression levels of ABCA1 gene were detected by quantitative PCR.2.Total DNA was extracted by No.DP332 TIANamp Blood DNA kit of Beijing Tiangen Company.We used Methyl Target sequencing to analyze DNA methylation at Cp G loci in promoter region of ABCA1 gene.3.Rs2246298、rs1800976、rs1800977、rs2437817、rs539621172、rs2740483 were genotyped by Kompetitive Allele-Specific PCR(KASP)technology.Then we compared the differences of DNA methylation levels and mRNA expression levels of ABCA1 gene among different genotypes.Results: 1.The mRNA expression levels of ABCA1 in the cases were significantly higher than that in controls(P<0.05).2.Compared with the controls,the DNA methylation levels of ABCA1 gene promoter in the cases were significantly lower than that in controls(P<0.05).Sex stratification analysis have found no significantly differences between the two groups(P>0.05).3.Rs2246298 and rs1800977 were significantly correlated with the methylation level of Cp G loci(P<0.05).4.Rs1800976,rs1800977,rs2246298,rs2437817,rs2740483 were not significantly correlated with the expression of ABCA1 gene(P>0.05).Part2: The effects of DNA methylation on promoter region of ABCA1 gene expression in foam cellsMaterials: The human monocyte THP-1 cell lines and THP-1 derived foam cellsMethods: 1.Total RNA in cells was extracted by cell total RNA extraction kit of Beijing Tiangen Company.All processes according to the manufacturer’s instructions.Total RNA was reversely transcribed into complementary DNA(c DNA)by Fast King RT Kit of Beijing Tiangen Company.The expression levels of ABCA1 gene were detected by quantitative PCR.2.Total DNA in cells was extracted by cell DNA kit of Beijing Tiangen Company.We used Methyl Target sequencing to analyze the levels of DNA methylation at Cp G loci in promoter region of ABCA1 gene.3.The total cell protein was extracted by the cell tissue protein extraction kit.And the protein was quantitatively detected.Results:1.The mRNA expression levels of ABCA1 in THP-1 cells were significantly lower than that in foam cells.2.Compared with the THP-1 cells,the DNA methylation level of ABCA1 gene promoter in the foam cells has no significant differences(P>0.05).3.Compared with the THP-1 cells,the DNA methylation levels of Cp G1、Cp G11 and Cp G16 of ABCA1 were significantly lower than that in foam cells(P<0.05).4.The protein expression levels of ABCA1 in the foam cells was significantly higher than that in THP-1 cells(P<0.05).Conclusions: 1.Hypomethylation in the promoter region of ABCA1 gene is closely related to the occurrence of ischemic stroke.2.Rs2246298 and rs1800977 in promoter region of ABCA1 gene are significantly correlated with the methylation levels of Cp G loci.And it is a potential marker of ischemic stroke.