ncRNAs靶向调控PI3K/Akt/mTOR通路参与胃癌遗传易感及其机制研究

Gastric cancer is a common malignant tumor existing individual susceptibility, but its etiology and mechanism is not very clear. We have found that the genetic variation of key genes from PI3K/Akt/mTOR pathway was involved in the development of gastric cancer. Studies have shown that noncoding RNA (ncRNA) and its genetic variation can regulate PI3K/Akt/mTOR pathway involved in the development of tumor, but it has not yet been reported in gastric cancer. In this study, ncRNAs targeting PI3K/AKT/mTOR pathway will be screened by high-throughput detection, systematic bioinformatics analysis and multi-stage population validation. The molecular epidemiology methods are used to evaluate the relationship between single nucleotide polymorphism (SNP) of ncRNAs gene and gastric cancer risk and its possible interaction in multi-stage population. The methods of double luciferase reporter gene, gene editing, chromosome conformation capture and nude mice bearing tumor will be used to explore the genetic and epigenetic mechanisms of risk SNPs and its host ncRNAs by regulating PI3K/AKT/mTOR pathway in the development and progression of gastric cancer. The study will further reveal the pathogenesis of gastric cancer, and provide theoretical basis and important reference for the future implementation of individual prevention, intervention and treatment on gastric cancer.

胃癌是常见的恶性肿瘤，存在个体易感性，但其病因和机制尚不很清楚。申请者前期发现，PI3K/Akt/mTOR通路关键基因遗传变异参与胃癌发生发展。研究表明，非编码RNA（ncRNA）及其遗传变异可调控PI3K/Akt/mTOR通路参与肿瘤发生发展，但尚未见与胃癌的相关报道。本研究拟采用高通量检测、系统生物信息学分析、多阶段人群验证，筛选靶向调控PI3K/AKT/mTOR通路的ncRNAs；运用分子流行病学方法，在多阶段人群中评价ncRNAs基因的单核苷酸多态性（SNP）与胃癌发病风险的关联性强度及其可能的交互作用；采用双荧光素酶报告基因、基因编辑、染色体构象捕获、裸鼠荷瘤等实验，探讨易感位点及其宿主ncRNAs调控PI3K/AKT/mTOR通路参与胃癌发生发展的遗传和表观遗传调控机制。该研究将进一步揭示胃癌发病机理，为今后实施胃癌的个体化预防、干预和治疗等提供理论依据和重要参考。

非编码RNA（ncRNA）及其遗传变异可调控PI3K/Akt/mTOR通路参与肿瘤发生发展，但与胃癌的相关研究鲜有报道。本课题拟探讨易感位点及其宿主ncRNAs调控PI3K/AKT/mTOR通路参与胃癌发生发展的遗传和表观遗传调控机制。在大样本的病例-对照研究中，我们明确了PI3K/Akt/mTOR 通路基因中潜在功能性 SNP 与 GC 风险的关联，并通过千人基因组计划数据库筛选出了4个候选单核苷酸多态性（即PIK3R1中的rs3730089和rs375668、PIK3R3中rs7536272和AKT1中的rs1130233）。Logistic回归分析结果表明rs7536272与胃癌的易感性加性模型显著相关（OR=1.16，95%CI=1.03–1.30，P=0.012）。转染两株胃癌细胞系SGC-7901和BGC-823重组质粒含有rs7536272 A等位基因或G等位基因。我们发现与对照组相比，rs7536272 G等位基因显著增加两种细胞系中的rs7536272 A等位基因（BGC的P=0.021和P=0.019-分别为823和SGC-7901细胞系）。此外，还进行了qRT-PCR检测评估了rs7536272基因型对GC组织中PIK3R3表达的影响，eQTL结果表明携带rs7536272 AG/GG基因型的人的PIK3R3显著较高与AA基因型的水平相比（P=0.025），其为与荧光素酶测定结果一致。此外，我们通过TCGA胃癌样本（375 Tumor vs 32 Normal）中共有1369个差异表达lncRNA（| logFC |> 1, P < 0.05, FDR< 0.05 ）。筛选出共有20个差异表达lncRNA与目标通路mRNA共表达相关。其中，lncRNA RP11-6O2.3差异表达，且与PI3K通路基因表达相关。进一步明确了lncRNA RP11-6O2.3上的位点rs260118与胃癌发生风险相关，并以该lncRNA表达的调控量进行了一系列细胞表型实验，验证了其对生物行为作用的影响。本研究探讨了易感位点及其宿主ncRNAs调控PI3K/AKT/mTOR通路参与胃癌发生发展的遗传和表观遗传调控机制，进一步揭示了胃癌发病机理，为今后实施胃癌的个体化预防、干预和治疗等提供理论依据和重要参考。