To Express Explore Deacetylase SIRT1 Gene And Its Associated Research And Chr6p21.32 Area Of ​​SNP Rs9268402 Polymorphism And Coronary Heart Disease And Coronary Heart Disease Pathogenesis

Part1:Association of gene expression, genetic variants of SIRT1with coronary artery diseaseBackground and Objectives:As a complex disease, the pathogenesis of coronary artery disease （CAD） was considered to be related with genetics factors.Recent studies demonstrated protective roles of silent mating type information regulation2, S. cerevisiae, homolog1（SIRT1） in vascular biology and atherosclerosis in cell and animal models.Polymorphisms related to theSIRT1gene have been reported to be associated with senescence, obesity, diabetes mellitus, Alzheimer’s disease, etc. But the association of SIRT1gene expression, genetic variants with CAD has not been studied systematically. Here, the relationship between mRNA expression level of the SIRT1gene and single nucleotide polymorphisms （SNPs） was investigated in the context of CAD, and the study was aimed to provide a new insight into the genetic control of SIRT1gene to the pathogenesis of CAD.Methods:1. The whole genome expression microarray （Illumina HT12v3） was conductedin peripheral blood mononuclear cells （PBMCs） in CAD group （18cases） and control group （6cases）.To analyze the mRNA expression level of the SIRT1gene and replicate the microarray data, real-time PCR replication was performed in PBMCs sample in an independent cohort [CADgroup:58cases, including28acute myocardial infarction （AMI） patients and30unstable angina （UA）;control group:175cases].2. SNPs were studied in10kb up-/downstream region of SIRT1gene. Then we analyzed the SNPs in linkage disequilibrium （LD） blocks via3bioinformatics’ websites to predict potential regulatory SNPs, and four candidates were selected to be further studied.3. The selected four SNPs were analyzed in previous CAD GWAS microarray data of the Chinese Han population. The association result was verified by Taqman allelic discrimination assay system in an independent cohort （angina group:1,235cases; control group:905cases）.4. Expression level of the SIRT1gene was analyzed by real-time PCR and the four SNPs were genotyped by Taqman probe in PBMCs from healthy subjects （n=229）. The relationship between SIRT1mRNA expression and genotypes of four possible regulatory SNPs （rs12778366, rs3758391, rs2273773and rs4746720） was evaluated. Results:1. In the microarray study, mRNA expression of the SIRT1gene, as one of the most significantly differential expression genes, was significantly lower in patients with ACS and CAD groupscompared with the healthy subjects. Replication showed that the expression of SIRT1inACSgroup were42.22%of that in control group. Specifically, the SIRT1expression of AMI and UA group among ACS patients were respectively37.78%,47.78%. The microarray results were verified in an independent cohort.2. Via the analysis of related database and bioinformatics’websites, four SNPs, rs12778366, rs3758391, rs2273773and rs4746720, were selected to proceed to the further studies.3. The allele C of rs12778366was found to associate with occurrences of Angina in CAD GWAS microarray data （OR=1.25, P=0.032）, but this result was not verified in an independent in angina case-control cohort （OR=0.96, P=0.66）.4. In healthy subjects, SIRT1mRNA expression levelwasfirst found to be significantly lower in minor allele C of rs3758391. The minor allele C of rsl2778366was associated with higher BMI, and the minor allele C of rs2273773was related to lower BMI, fastingglucose and higher diastolic blood pressure.Conclusions:1. Our study explored the relationship between the expression of SIRT1mRNA and CAD, and confirmed that SIRT1mRNA expression was reduced in CAD patients intwo CAD case-control cohorts via two techniques, whole genome expression microarrayand real-time PCR, suggesting that the gene SIRT1might confer a positive role against CAD in cohort study.2. Four potentialregulatory SNPs were selected from the non-coding SNPs and synonymous variants related to the SIRT1genes.3. Our study did not found the association between SIRT1related SNPs and CAD. The result suggested that SIRT1related SNPs were not related with CAD, or need larger sample size to verify.4. The association between these variants and the SIRT1mRNA expression was conducted andour study first found that SIRT1expression was lower in the minor allele C of rs3758391, which had been reported to be in the binding sites of p53to the upstream of the SIRT1gene. The fact that the change of SIRT1mRNA is associated with SNPs nearby provides novel insights into CAD pathogenesis and new targets of prevent and treatment of CAD. Part2:Study on the relationship between susceptible SNP rs9268402at chromosome6p21.32and pathogenesis of coronary artery diseaseBackground and Objectives:Genomewide association studies （GWAS） have led to the identification of more than fifty loci of atherosclerotic cardiovascular disease, and it has been into the era of discovering the patho-physiological mechanisms underlying these loci. Here, we tried to explore the regulatory mechanism of risk SNP rs9268402in6p21.32locus of coronary artery disease （CAD） in Chinese Han population, and then provide a new target of prevention and treatment of CAD.Methods:1. According to previous studies, the expression of transcripts adjacent to the6p21.32locus of coronary artery disease might be affected by the genotypes of rs9268402. Two adjacent protein coding genes C6orf10and BTNL2, one non-protein coding gene HCG23, and one long noncoding transcript of unknown function, designated TCONS₀0012469, were selected as potential target genes for rs9268402. Expression level was analyzed by real-time PCR and rs9268402was genotyped by Taqman probe in peripheral bloodmononuclear cells （PBMCs） sample （n=78）.2. The potentialregulatory functions of rs9268402and its proxy SNPs, rs3817973and rs9268473, which were in strong linkage disequilibrium （LD） with rs9268402（both of the two SNPS,r2=0.74）, were predicted in the Encyclopedia of DNA Elements （ENCODE） database, RegulomeDB bioinformatics’website and HaploReg v2bioinformatics’websites,3. In that rs3817973was predicted to be a potential regulatory SNP and in strong LD with rs9268402, the genomic fragment of the predicted regulatoryregion （chr6:32360556-32362973） where rs3817973resided in wascloned into the pGL3promoter vector. The recombinant plasmids were transfected into HEK293T cells and then dual luciferase reporter activity assay was performed to detect allele specific transcriptional activation activity.4. Electrophoretic mobility shift assay （EMSA） was conducted using nucleus extract ofhuman umbilical vein endothelial cells （HUVEC）. Then multiplexed competitor EMSA （MC-EMSA） was applied to search for the specific transcription factors that bind to allele A of rs3817973.Results:1. TranscriptTCONS₀0012469were significantly increased in carriers of the risk allele G of rs9268402, suggesting lncRNATCONS₀0012469might be a target gene of rs9268402.2. Rs3817973, in strong LD with rs9268402, was found in a rich region ofH3K27Ac and H3K4Mel histone marks, DNAasel hypersensitivity and binding sites of transcription factors in HUVEC. What is more, rs3817973locates in the first exon of lncRNA TCONS00012469.3. Theluciferase reporter activity assay showed that the transcriptional activity of the two alleles was stronger than empty vector, whileit did not be significant between the two alleles.4. EMSA demonstrated that allele A of rs3817973had greater binding affinity of nuclear protein than allele G. MC-EMSA showed that five transcription factors might be specific to bind allele A of rs3817973. They were androgen receptor, CREB binding protein, interferon-gamma activated sequence, interferon-stimulated response element and interferon regulatory factor1.Conclusions:1. Our studies found that the major allele G of rs9268402, in6p21.32risk regionof coronary artery disease, was associated with the high expression level of lncRNA TCONS₀0012469, suggesting that it might be one of the target genes of rs9268402.2. Bioinformatics’ predictions indicated that rs9268402might function through one of its proxy SNP rs3817973as an enhancer, which suggests that rs3817973, resided in the first exon oflncRNA TCONS₀0012469, mightregulatethe transcription of the lncRNA or other target genes.3. The luciferase reporter activity assay confirmed that the predictedregion, encompassing rs3817973, could enhance gene transcription.4. EMSA found the minor allele A of rs3817973bind five transcription factors. Therefore, rs3817973might regulate the expression of its target genes by binding allelic different transcription factors and then participates inpathogenesis of CAD.