全基因组CpG-SNPs与早发胃癌发病风险及其机制研究

Early-onset gastric cancer (EOGC) is a subtype of gastric cancer in patients younger than 45 years old and the exact mechanism is not yet clear. Previous studies and our study revealed that CpG-SNPs play important roles in the development and progression of diseases by genetic and epigenetic regulation. However, the role of CpG-SNPs in EOGC development was never investigated. We were the first time to use high-throughput detection technology and GWAS data to screen 27 CpG-SNPs related to EOGC susceptivity. We suspect that CpG-SNPs can change the function of key genes by modulating DNA methylation, and participate in EOGC development. We plan to perform case-control studies to analysis the association between EOGC risk and CpG-SNPs. Besides, we will conduct the methods of CRISPR/Cas9, ChIP, EMSA and 3C to investigate the mechanism of CpG-SNPs involved in the development and progression of EOGC. Finally, risk prediction model will be constructed based on our EOGC clinical cohort to evaluate the value of CpG-SNPs as potiential biomarkers. This study will systematically clarify the regulation mechanism of CpG-SNPs involved in EOGC development. It will provide important theoretical basis and practical guidance for personalized prevention and treatment.

早发胃癌是指患者发病年龄≤45岁的一类胃癌，存在明显个体易感性，但机制不清。文献及本课题组研究表明，DNA甲基化相关SNPs（CpG-SNPs）通过调控遗传（主要是SNPs）和表观遗传（如DNA甲基化）互作而参与疾病的发生发展，但未见早发胃癌的研究报道。本课题组前期基于甲基化芯片、早发胃癌GWAS以及公共数据库筛选出27个与早发胃癌发病风险相关的CpG-SNPs。我们假设：CpG-SNPs通过改变周围DNA甲基化状态，影响关键基因功能而参与早发胃癌发生发展。本项目拟运用多阶段病例-对照研究，分析候选CpG-SNPs与早发胃癌发病风险及预后关联性；采用CRISPR/Cas9、EMSA、染色体构象捕获（3C）等方法，探讨CpG-SNPs的生物学功能；构建风险预测模型，在早发胃癌临床专病队列中评价其作为潜在生物标志物的价值。该研究将为阐释早发胃癌发病机制及今后个体化防治提供重要理论依据。

CpG-SNPs是将遗传（如SNPs）和表观遗传（如DNA甲基化）有机结合的一种形式，并被发现参与了包括恶性肿瘤在内的多种复杂疾病的发生发展，本课题组将从CpG-SNPs入手，基于全基因组水平，鉴定出与早发胃癌风险相关的易感位点和易感基因；采用细胞恶性表型实验、免疫荧光原位杂交技术、双荧光素酶报告基因实验及生物信息学方法等揭示易感基因参与胃癌发生发展的作用机制。结果显示CpG-SNP rs9891656 T等位基因是早发胃癌发病的风险等位基因（OR=1.74，95%CI=1.44-2.10，Pmeta=9.24×10-9），rs9891656能够显著影响SUZ12P1的启动子区的甲基化水平，并一定程度下调该基因的表达水平，最终影响早发胃癌的发病风险。SUZ12P1在胃癌组织中呈现高表达，当干扰其表达水平时，胃癌细胞的增殖和克隆能力降低、侵袭和转移能力受到抑制。SUZ12P1在细胞核内和细胞质中均有表达，其在细胞核可与其同源基因SUZ12结合；在细胞质中则通过扮演“内源性竞争RNA”（competing endogenous RNA，ceRNA）的角色与其同源基因SUZ12竞争性结合miRNA-200b-3p和miRNA-200c-3p从而促进SUZ12表达。SUZ12P1与SUZ12的表达在胃癌组织中呈现显著的正相关。课题组还基于胃癌全基因组关联研究（GWAS）数据，采用最小绝对收缩和选择算子（LASSO）和遗传算法结合的策略，筛选与胃癌发病风险密切相关的SNPs位点并构建遗传风险预测模型，利用早发胃癌人群数据对其预测早发胃癌发病风险的效能加以验证。此外基于甲基化芯片和亚硫酸氢盐测序，课题组分析了DNA甲基化位点与早发胃癌的关联性，DNA甲基化及CpG-SNPs在胃癌发生发展中的作用。本研究通过一系列分子生物学技术探讨了早发胃癌易感性相关的CpG-SNPs及其基因的生物学功能，为进一步揭示早发胃癌发病机制提供理论依据。