Single Nucleotide Polymorphism And The Mirna Regulatory Mechanism Of Carm1 Gene Expression And Its Role In Coronary Heart Disease Research

Objective:The study was designed to clarify the molecular regulation mechanism of coactivator-associated arginine methyltransferase1(CARM1) expression and explore its function in coronary artery disease (CAD). The attempt to uncover the function and mechanism of CARM1gene might provide a new target of prevention and treatment of CAD.Methods:1) The mRNA and protein expression levels of CARM1in PBMCs between CAD and control groups were detected with real-time PCR and Western blot;2) The potential functional SNPs in different linkage disequilibrium (LD) blocks were screened by integrating evidence of both HapMap project database and Encyclopedia Of DNA Elements (ENCODE) functional database; the relationship between these SNPs and CARM1mRNA expression levels in234healthy subjects was analyzed; the biological functional studies of the SNPs related with CARM1expression were conducted as follows:dual luciferase reporter activity assay to detect allele specific transcriptional activation activity; electrophoretic mobility shift assay (EMSA), multiplexed competitors EMSA (MC-EMSA), supershift-EMSA and ChIP to screen and determine the transcription factors specifically binding to different alleles, respectively in vitro and in vivo;3) Based on bioinformatics prediction of target miRNAs of CARM1, the miRNA expression level was detected in CAD cases and controls with real-time PCR; co-transfection of luciferase reporter gene containing CARM13’-UTR with or without seed mutations and miRNA mimics was performed to determine the binding of miRNA to CARM13’-UTR; the regulation of mRNA and protein expression level of CARM1with overexpression or knockdown of miRNA was detected;4) To explore the regulation of homocysteine (Hcy) by CARM1, we conducted the association study of the functional variant in CARM1with plasma Hcy levels and measured the concentration of Hcy in cell supernatants and lysates with ELISA after overexpression human CARM1plasmid;5) Association study of the functional variant in CARM1with CAD in1010cases and3998controls was conducted, and the effect of the minor allele on CAD risk was examined in an additive genetic model.Results:1) Compared with the control group, the mRNA and protein expression level of CARM1in PBMCs of acute coronary syndrome (ACS) group increased3.9fold and1.5fold, respectively;2) We seletcted4potential functional variants based on the evidence of LD analysis and ENCODE database,3of which were related with CARM1expression levels in healthy population. These3SNPs were rs12460421(A/G), rs117569851(T/C) and rs4804544(A/G), and the last two SNPs were completely linkage disequilibrium. The luciferase reporter activity assay showed that the transcriptional activity of rs12460421-rs117569851haplotype GC and AT was2.0fold and1.7fold of that of haplotype GT, repectively; EMS A demonstrated that A allele of rs12460421had greater binding affinity of nuclear protein than G allele, and C allele of rs117569851had greater binding affinity of nuclear protein than T allele; MC-EMSA showed that Egr-1might bind to both of the SNPs; supershift-EMSA and ChIP assay certified the identification of Egr-1binding to the two SNPs with allele specific binding affinity;3) miR-15a and miR-16was predicted to target CARM1using bioinformatics method; miR-15a expression level in PBMCs was decreased by37%in ACS cases compared with controls, while miR-16was upregulated in ACS cases; after co-transfected of luciferase reporter gene containing CARM13’-UTR with or without seed mutations and miR-15a mimics, the results of dual luciferase reporter assay system showed that miR-15a binded to both of the seeds of miRNA binding sites in CARM13’-UTR; CARM1expression was upregulated by the knockdown of miR-15a, however it was not downregulated by the overexpression of miR-15a;4) The functional variant rs117569851in CARM1was associated with plasma Hcy levels in a healthy population involving406subjects (P=0.02), and the Hcy levels decreased2.16μmol/L per minor allele T;5) Genetic analysis did not find the association of rs117569851with CAD (P=0.053, adjusted by age, sex and BMI).Conclusions:1) The mRNA and protein expression of CARM1in ACS patients increased compared with controls, which indicated that CARM1might play a role in CAD pathogenesis;2) The minor allele T of rs117569851locating in CARM1promoter downregulated the expression level of CARM1gene through lower binding affinity of transcription factor Egr-1, decreased the plasma Hcy level, and potentially reduced the risk of CAD;3) The decreased expression of miR-15a in PBMCs of CAD patients might reduce the inhibition effect on mRNA degradation and protein translation of CARM1, inducing CARM1expression upregulated;4) Association studies showed that CARM1might play a role in CAD pathogenesis through regulating Hcy metabolism. Objectiversl059611, located in3’-UTR of LPL, is associated with plasma lipid concentrations in GWAS, but its biological function is unclear. The study is designed for tentative exploration of its effect on transcriptional activity and transcription factor affinity.Methods1) The genomic sequence containing rs1059611was inserted into the downstream of the reporter gene in pGL-3promoter vector to construct a recombinant plasmid; the effect of rs1059611on transcriptional activity was illustrated with dual-luciferase reporter activity system;2) Incubation with nuclear extract from human visceral adipose tissue and probes containing rsl059611(C/T) sequence, followed by electrophoretic mobility shift assay (EMSA) was conducted to compare the differences of shift bands formed by probes and nuclear protein between two alleles; competitor EMSA was carried out with pre-incubation with competitors of allele C and T with nuclear extract respectively and adding biotin labeled probes later, to observe whether the shift bands vanish.Results1) The transcriptional activity was enhanced by sequence containing rsl059611, and increased significantly after major allele T converting to minor allele C (0.69vs.1.00);2) the greyscale of shift bands formed by probes of allele T for rsl059611binding with nuclear extract of adipose tissue was higher than allele C; the discrepancy of shift bands between two alleles with nuclear extract of adipose tissue from individual A was greater than that from individual B; the shift bands were completely abolished by pre-incubation with200fold competitor probes.ConclusionThe transcriptional activity was increased after major allele T converting to minor allele C; the affinity of major allele T binding to transcription factor was greater than that of minor allele C. It declared that rsl059611might have biological functions of affecting transcriptional activity.