功能性遗传变异调控mTOR复合体组成成分基因表达及影响胶质瘤发病与转归的分子机制研究

Previous studies on genetic variation and cancer usually focused on gene coding region and adjacent regulatory region. However, the results of genome-wide association studies (GWAS) and expression quantitative trait locus (eQTL) analyses conducted in recent years indicate that genetic variation located in intergenic region and intron, such as genetic variation in enhancer and eQTL, is also of importance. As a pivotal component of PI3K/AKT/mTOR signal transduction pathway, mTOR complex plays an important role in the genesis and development of glioma. Our preliminary experiment showed that there is an enhancer located in the intron of MAPKAP1 which is a component of mTORC2. Furthermore, a genetic variation rs473426 in the enhancer can influence the binding ability of corresponding transcription factors and change the regulatory activity of the enhancer, and thereby associatied with risk of glioma. Based on these results, this project aims to investigate the association of genetic variations in enhancers, eQTLs and adjacent regulatory regions of mTOR complex genes, which might be involved in the regulation of mTOR complex genes expression, with the development and prognosis of glioma through a multicenter and multistage association study. A series of biological assays, such as EMSA and chromatin conformation capture, will be used to investigate the function of these genetic variations. The results of this project will provide novel strategies and theoretical foundations for the prevention and clinical therapeutic decision of glioma.

以往关于遗传变异与肿瘤的研究多聚焦于基因编码区及其邻近调控区。然而，近年来的全基因组关联研究（GWAS）及表达数量性状位点（eQTL）分析表明，位于基因间隔区和内含子区的遗传变异（如增强子遗传变异和eQTL）亦有重要作用。mTOR复合体作为PI3K/AKT/mTOR信号传导通路的关键枢纽，与胶质瘤的发生发展关系密切。预实验发现，mTOR复合体mTORC2的组分MAPKAP1基因内含子区存在一个增强子，其中的遗传变异rs473426可影响转录因子的结合，改变增强子的调控活性，并与胶质瘤的发病相关。据此，本研究拟通过多中心多阶段的关联研究，系统分析与mTOR复合体组成成分基因表达调控相关的增强子遗传变异、eQTLs及基因邻近调控区遗传变异与胶质瘤发病及转归的关系，并运用EMSA和染色质构象捕获等一系列生物学实验，阐明这些遗传变异的作用机制，为胶质瘤的预防和临床治疗决策提供新的思路和理论依据。

胶质瘤是最常见的原发性中枢神经系统恶性肿瘤，预后差。mTOR复合体在胶质瘤的发生发展中发挥重要作用。研究遗传变异与肿瘤发生发展的关系是科学家多年来热衷的工作。然而，以往的研究较少涉及基因间隔区等非编码区。本研究旨在分析mTOR复合体组分基因的增强子遗传变异、表达数量性状位点及基因邻近调控区的遗传变异与胶质瘤发生发展的关系。本研究首先分析了mTOR复合体组分基因的增强子元件、表达数量性状位点及基因邻近调控区的遗传变异，并筛选出我国南方汉族人群常见遗传变异作为候选遗传变异。然后运用胶质瘤病例/对照研究、生存分析、双荧光素酶报告基因实验等方法，探讨候选遗传变异与胶质瘤发病及转归的关系及分子机制。结果表明mTORC1重要组分RPTOR基因的表达数量性状位点rs7502563以显性模型方式显著降低胶质瘤发病风险[比值比(OR)=0.59，95%可信区间(CI)=0.38–0.89, P=0.0123]，分层分析显示rs7502563与胶质瘤发病的相关性在女性、老年及高级别胶质瘤中更为显著，结果分别为(OR=0.40, 95%CI=0.20–0.80, P=0.0091)，(OR=0.47, 95%CI=0.26–0.86, P=0.0135)和(OR=0.45, 95%CI=0.27–0.77, P=0.0031)。分层分析还发现，随着胶质瘤级别的升高，OR逐渐降低。我们还发现rs7502563G等位基因在多个不同人群中均与RPTOR基因高表达显著相关。以上结果均表明mTOR复合体组分基因表达数量性状等遗传变异可改变相应基因表达水平，从而影响胶质瘤的发生发展。