COL6A2基因3’-UTR区域功能单核苷酸多态性位点rs1044598参与miR-4252调节中国汉族人群房间隔缺损发

发布时间：2018-06-23 23:28

本文选题：先天性心脏病 + 易感性　；参考：《南京医科大学》2016年硕士论文

【摘要】：目的：文献报道显示干预小鼠体内COL6A2基因的表达可致子代小鼠出现先天性房间隔缺损的病理表型,揭示COL6A2基因的表达水平变化与房间隔缺损的发病相关,但具体的调节机制尚不清楚。非编码RNA (microRNA, miRNA)是一类序列较短,有着高度保守性的RNA,可通过与靶基因的3'-UTR (3'-Untranslated Region)区域特异结合,调节基因的转录活性。而靶基因3’-UTR区域单核苷酸多态性变异(single nucleotide polymorphism, SNP)可能影响miRNA与之结合,导致靶基因表达的变化。本实验旨在探讨COL6A2基因上3'-UTR区域SNPs 是否影响 miRNA与之结合,从而在调节其在先天性房间隔缺损的发生中发挥重要作用。方法：通过查询Pubmed以及Hapmap数据库搜索COL6A2基因3'-UTR区域中国汉族人群最小等位基因频率大于0.05的SNP位点,结合各SNP位点之间的连锁不平衡反应,初步筛选拟研究位点。随后在本研究小组前期先天性心脏病间隔缺损全基因组关联研究数据库(Genome Wide Association Studies)中查找上述位点的数据信息,选取在先天性房间隔缺损中关联P值小于0.1的SNPs,再使用miRNA-SNP功能预测网站进行功能预测,选择预测对结合能力有影响的SNPs作为功能研究位点。将构建成功的不同miRNA的模拟物(mimic)及COL6A2基因3'-UTR序列不同SNP基因型的质粒共同转染入HEK293T细胞系、H9C2细胞系以及SD乳鼠原代细胞系进行荧光素酶报告基因实验。共分为四组：A.野生型质粒+无miRNA mimic; B.野生型质粒+miRNA mimic; C.突变型质粒+无miRNA mimic; D.突变型质粒+miRNA mimic。探讨miRNA与COL6A2基因3’-UTR区域的结合反应,以及对COL6A2基因表达水平变化的影响。结果：通过查询Pubmed以及Hapmap数据库,COL6A2基因3'-UTR区域共搜索到5个中国汉族人群最小等位基因频率大于0.05的SNP位点,分别为rs559,rs1044598, rsl 1554666, rs7717和rs73382475。通过对先天性心脏病间隔缺损GWAS数据库的查询,发现仅rs559和rs1044598位点的变异可能与先天性房间隔缺损发病风险相关。功能预测结果显示,rs559不可调节miR-338-3p与COL6A2的结合,rs1044598可调节miR-4252与COL6A2的结合。以下为荧光素酶报告基因实验P值：miR-4252与rs1044598质粒共转染组,D组与B组相比,HEK 293T(P=0.013), H9C2 (P=0.012),原代心肌细胞(P=0.009) 。 miR-338-3p与rs559质粒共转染组,A组与C组相比,HEK293T(P=0.158), H9C2 (P=0.342),原代心肌细胞(P=0.054)。结论：COL6A2基因3'-UTR区域rs1044598位点可能与先天性房间隔缺损的发病风险相关,相对于携带野生TT基因型的人群,AA基因型显著减少了36%的患病风险。miR-4252可以与COL6A2的3'-UTR区序列发生有效结合,与野生型TT基因型相比,变异型AA等位基因可使miR-4252结合更稳定,并进一步抑制COL6A2的转录活性,降低该基因表达水平,从而减少先天性房间隔缺损的发病风险。而miR-338-3p与COL6A2的3'-UTR区序列不能发生有效结合,从而无法调控COL6A2的表达水平。
[Abstract]:Objective: it has been reported that the expression of COL6A2 gene in mice can induce the pathological phenotype of congenital atrial septal defect in offspring, and the expression level of COL6A2 gene is related to the pathogenesis of atrial septal defect. But the specific regulation mechanism is not clear. Non-coding RNA (miRNA) is a kind of short sequence, highly conserved RNAs, which can regulate the transcriptional activity of the gene by specifically binding with the target gene's 3na-UTR (3- untranslated region) region. The single nucleotide polymorphisms (SNPs) in the target gene 3nc-UTR region may affect the binding of miRNAs and lead to the change of target gene expression. The aim of this study was to investigate whether SNPs in the 3H-UTR region of COL6A2 gene affect miRNA binding and thus play an important role in regulating the occurrence of congenital atrial septal defect. Methods: by querying Pubmed and Hapmap database, we searched for SNP loci in Chinese Han population with minimum alleles frequency greater than 0.05 in COL6A2 gene 3na-UTR region. Combined with linkage disequilibrium reaction among SNP loci, we preliminarily selected the sites to be studied. We then searched the Genome wide Association Studies database for these sites in our team's Genome wide Association Studies database for the early stage of congenital heart disease septal defect. SNPs with P value less than 0.1 in congenital atrial septal defect were selected, and then miRNA-SNP function prediction site was used to predict the function of SNPs. SNPs, which had influence on binding ability, were selected as functional research sites. The plasmids with different SNPs of (mimic) and COL6A2 gene were co-transfected into H9C2 cell line of HEK293T cell line and primary cell line of SD rat for luciferase reporter gene experiment. It was divided into four groups: A. Wild type plasmid without miRNA mimic. B. Wild type plasmid miRNA mimic; C. The mutant plasmid has no miRNA mimic.D. Mutant plasmid miRNA mimic. To study the binding reaction of miRNA with COL6A2 gene 3- UTR region and the effect of miRNA on the expression level of COL6A2 gene. Results: five SNPs with minimum alleles frequency greater than 0. 05 were found in Pubmed and Hapmap database. The minimum alleles were rs559, rs1044598, rsl 1554666, rs7717 and rs73382475, respectively. Only rs559 and rs1044598 loci were found to be associated with the risk of congenital atrial septal defect (CHD) by querying the Gwas database of congenital heart disease septal defect (CHD). The results of functional prediction showed that the combination of rs559 unadjustable miR-338-3p and COL6A2 could be adjusted by the combination of miR-4252 and COL6A2. The following is the experiment of luciferase reporter gene P value: miR-4252 and rs1044598 plasmid cotransfection group compared with group B: HEK293T (P0. 013), H9C2 (P0. 012), primary cardiomyocytes (P0. 009). MiR-338-3p co-transfected with rs559 plasmid group A compared with group C, HEK293T (P0. 158), H9C2 (P0. 342), primary cardiomyocytes (P0. 054). Conclusion the rs1044598 locus of the 3-UTR region of the 1: COL6A2 gene may be associated with the risk of congenital atrial septal defect. Compared with the population carrying the wild TT genotype, the AA genotype can significantly reduce the risk of the disease by 36%. MiR-4252 can be effectively combined with the 3- UTR region sequence of COL6A2. Compared with the wild-type TT genotype, the mutant AA allele could make miR-4252 binding more stable, further inhibit the transcription activity of COL6A2 and decrease the expression level of the gene, thus reducing the risk of congenital atrial septal defect. However, miR-338-3p could not combine with COL6A2 3-UTR region effectively, which could not regulate the expression level of COL6A2.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R541.1

【相似文献】