错配修复基因剪接相关SNPs与胃癌易感性及其机制研究

Gastric cancer (GC) characterized by the inter-individual variation of susceptibility is caused and promoted by multi-gene and multi-factor in multi-stage. In this process, the DNA mismatch repair (MMR) known as one of the crucial DNA damage repair pathways may play an important role, because of its functions in preventing deleterious effects from carcinogens and safeguarding the integrity of the genome. The single nucleotide polymorphisms (SNPs) in MMR genes may be associated with differences in the MMR capacity to repair DNA damage, and so this can influence an individual's susceptibility to cancer. However, the pathogenic molecular mechanisms of these SNPs are still unclear and their associations with GC risk are also needed to be explored. Our previous studies with limited samples found that some of the SNPs in MMR genes were associated with a significantly increased risk of GC. Moreover, function analyses based on bioinformatics showed potential effects from these SNPs on pre-mRNA splicing of MMR genes by interferences of splicing-regulatory elements. Therefore, based on previous evidences, a further study is planed to be developed as follows: ①the associations of SNPs in MMR genes with risk of GC will be analyzed in a large case-control study; ②the effects on pre-mRNA splicing from SNPs in MMR genes will be studied by an ex vivo functional assay based on splicing reporters and be further verified by an in vivo analyses of MMR genes expression and sequences in GC patients; ③splicing-regulatory elements associated with SNPs of MMR genes will be further identified to reveal the molecular mechanisms of splicing interferences due to those SNPs; ④The associations of MMR genotypes with clinical phenotypes of GC will be analyzed. This study will contribute to the knowledge on GC susceptibility and SNPs of MMR genes, and relevant pathogenic molecular mechanisms, which may provide important theory evidences for future individualized GC prevention and therapy.

胃癌的发生发展涉及多基因、多因素和多环节，且具有明显个体易感性。错配修复（MMR）基因是一组维持基因组稳定的关键性DNA 损伤修复基因，其单核苷酸多态性（SNPs）与肿瘤的发生发展密切相关，但至今确切机制仍不清楚。我们前期初步发现部分MMR基因SNPs使胃癌风险显著增加；生物信息学功能分析提示，相关SNPs可能影响剪接调控元件的活性，从而干扰MMR基因前体mRNA的剪接。本课题拟在前期研究基础上：①病例-对照研究验证MMR基因潜在剪接相关SNPs与胃癌易感性的关系；②利用剪接报告子从离体水平揭示相关SNPs对MMR基因前体mRNA剪接的影响，并通过患者MMR基因序列及表达分析从整体水平加以验证；③鉴定SNPs相关剪接调控元件以揭示SNPs干扰剪接的分子机制；④分析基因型-胃癌表型的关系。最终阐明MMR 基因SNPs与胃癌发生发展的关系及其功能机制，为胃癌的个体化预防和治疗提供重要理论依据。

胃癌的发病以环境与遗传因素的交互作用为特点，并具有明显的个体易感性。错配修复（MMR）主要修复DNA复制后的碱基错配，MMR异常引发微卫星不稳定（MSI），可继发基因组不稳定，导致突变率上升，患癌风险随之升高。单核苷酸多态性（SNPs）与个体对疾病的易感性有着密切联系。MMR基因SNPs影响肿瘤的发生发展，但与胃癌的关系及其机制仍不清楚。本研究对此开展了三个方面的研究：1）MMR基因SNPs与胃癌易感性的病例-对照研究；2）相关SNPs影响胃癌易感性的机制研究；3）相关SNPs与胃癌的临床相关性。研究纳入了423例病例和454例配对的正常对照。经生物信息学筛选，MLH1基因rs1800734、MSH2基因rs2303428、EXO1基因rs735943和TREX1基因rs11797等潜在功能性位点被纳入研究，结果发现：1）MLH1基因rs1800734多态G碱基显著降低胃癌的发病风险（GA or GG vs AA, OR = 0.72; 95% CI: 0.50-1.05; Ptrend for the G-allele = 0.029）；2）MMR基因SNPs风险基因型间有联合作用，随着携带风险基因型的增加，胃癌的风险亦显著增加（Ptrend = 0.039）；3）rs11797多态位点与饮绿茶和豆制品高摄取间存在交互作用，AG或AA基因型显著减低胃癌发病风险（P for interaction = 0.007 和 0.016）；4）MLH1基因rs1800734 AA较GG基因型中MLH1蛋白低表达的比例明显升高（25% vs 10%），差异接近统计学显著性（P = 0.078）；5）随着风险基因型的增加，MSI胃癌的风险显著增加，而微卫星稳定（MSS）胃癌的风险降低，提示MMR风险基因型的联合作用可能增加胃癌MSI表型而减少MSS表型；6）初步分析提示部分基因型可能与胃癌化疗疗效及预后有关，具有一定的临床转化应用前景。综上，本研究表明，MMR基因SNPs影响胃癌易感性，SNPs基因型和环境暴露因素间存在交互作用，风险基因型间存在联合作用并影响胃癌MSI表型，MLH1基因rs1800734 AA基因型可能影响MLH1蛋白表达；部分基因型与胃癌具有临床相关性。本研究揭示了MMR基因SNPs在胃癌中的作用，完善了胃癌的发病机制，为胃癌的个体化预防和治疗提供了理论依据