The Mechanism Of MiR-4252 Regulating Pathogenesis Of Atrial Septal Defect With SNP(rs1044598) In 3’-UTR Of COL6A2 In Han Chinese Populations

Objective:As reported, interrupting the expression of COL6A2 could result in atrial septal defect, which confirming the association between the expression of COL6A2 and the risk of congenital heart disease.However, the exact mechanism is unknown. MiRNAs are small, evolutionally conserved, non-coding RNA molecules which have been shown to negatively regulate gene expression by binding to the 3’-untranslated region.The ability of miRNA binding to gene could be affected by different genotype of SNP, which associates with risk of congenital heart disease. We aim at the asso-ciation between the SNP of COL6A2 and risk of atrial septal defect and the mecha-nism of miRNA regulating pathogenesy.Method:The SNPs with MAF(minimum allele frequency)>0.05 in Han Chinese populations and on 3’-UTR of COL6A2, were selected in database of Pubmed and Hapmap after excluding the high LD between them. Then the p-values of SNPs were checked in GWAS database to select the SNPs with p-value<0.1. Finally the function of the SNPs were predicted in the tool of miRNA-SNP to identify the targeting miRNA. Basing on the results, the plasmids(Psnp), the miRNA mimic and the negative control mimic were constructed to proceed luciferase reporter assay. The assay contained four groups:Group A—Plasmid(wild type)+Negative control mimic; Group B — Plasmid(mutated type) + Negative control mimic; Group C — Plasmid (wild type)+miRNA mimic; Group D—Plasmid(mutated type)+miRNA mimicResult:Based on the data from Pubmed and Hapmap, we select 5 SNPs:rs559, rs1044598, rsl 1554666, rs7717 和 rs73382475. Based on our previous GWAS of atrial septal defect, we found rs 1044598 and rs 559 may be new independent susceptibility locus of atrial septal defect in Han Chinese populations. Functional prediction show rs1044598 may regulate the binding ability of miR-4252 to COL6A2, meanwhile rs559 may not regulate miR-338-3p.The dual luciferase results as follows:After we co-transfected Prs1044598 into cells with miR-4252 mimic or negative control mimic,we found that compare group D with group B, luciferase expression significantly decreased(P values in groups: HEK293T, P=0.013; H9C2, P=0.012; primary myocardial cell, P=0.009). However, co-transfection Prs559 with miR-338-3p, luciferase expression changed little(P values in groups:HEK293T, P=0.158; H9C2, P=0.342; primary myocardial cell, P=0.054). However, the change of relative activity in primary myocardial cell was opposite to funtional prediction.Conclusion:Rs 1044598 may be new independent susceptibility locus of atrial septal defect in Chinese populations. To the rs 1044598:genotype AA can decrease the risk of atrial septal defect by 36% compared with genotype TT. To the rs 559:Neither of the two genotype can increase the risk of atrial septal defect.MiR-4252 could regulate COL6A2 and mRNA expression by binding to 3’-UTR of COL6A2. And the mutated genotype AA could increase the binding ability of miR-4252 to COL6A2. Thus it may inhibit the transcriptional activity to down-regulate the expression of COL6A2.