影响转录因子c-Myc结合的功能性遗传变异系统发掘及其与肝癌易感性的关系研究

Oncoprotein c-Myc can bind to the E-box DNA element (5'-CACGTG-3 ') and regulate many downstream target genes, which participate in the development and progression of hepatocellular carcinoma (HCC). Our previous studies showed that there are 34436 single nucleotide polymorphisms (SNP) located in the 12256 c-Myc binding sites in the HCC genome. Bioinformatics screening results revealed that there are 20 SNPs which locate in the E-box element and may interrupt binding of c-Myc to DNA. After genotyping 5 SNPs in 800 HCC cases and 800 age- and sex-matched healthy controls (the discovery cohort), we found that rs1180015 polymorphism, locating in a potential enhancer of the HNF1A gene, is significantly associated with increased HCC risk. Dual-luciferase reporter gene assays showed that rs1180015G→C change could lead to significantly reduced enhancer activities in both HepG2 and SMMC7721 cells. In this proposal, we are going to genotype the other 15 SNPs in the discovery cohort and validated the results in 1700 HCC cases and 1700 controls from two medical centers. The biological role of the HCC susceptibility-related SNPs will be examined by a set of biochemical assays, including EMSA and dual-luciferase reporter gene assays. After clarifying how the SNPs influence function of c-Myc in vitro, we will also detect expression of the c-Myc target genes in HCC tissue samples and normal liver tissue samples to elucidate the role of these polymorphisms in vivo. In summary, our study may provide a new strategy for in-depth study of the relationship between functional SNPs and HCC genetic susceptibility in the post-genome-wide association study era. Also, the identification of polymorphisms impacting c-Myc function would contribute to our understanding of the genetic basis of individual differences in c-Myc transcriptional regulation, and the molecular mechanism of hepatocarcinogenesis.

原癌基因c-Myc可通过特异结合靶基因E-box核心元件(5'-CACGTG-3')调控众多下游基因，从而参与肝癌的发生发展过程。我们的前期研究显示，在肝癌基因组12256个c-Myc结合位点中，存在34436个单核苷酸多态。生物信息学筛查表明，上述多态中，位于E-box元件、并可能导致c-Myc与DNA结合异常的共20个。对其中5个遗传多态基因分型后，我们发现，位于HNF1A基因潜在增强子中的rs1180015多态可影响增强子活性，并与肝癌易感性显著相关。本研究着眼于进一步揭示上述20个遗传多态中与肝癌易感性显著相关的位点，并通过EMSA和报告基因实验等一系列体内外实验，阐明这些遗传多态影响c-Myc对靶基因调控的分子机制。在"后GWAS时代"，本项目为深入研究功能性遗传变异与肝癌易感性的关系提供了一种新策略，并为进一步理解c-Myc转录调控个体差异的分子基础及肝癌发病机制提供了新思路。

肝癌是一种高发病率、高致死率的恶性肿瘤。虽然近年来的全基因组关联研究已揭示了大量疾病遗传易感单核苷酸多态性(SNPs)，功能性肝癌遗传致病因素的发现远未完成，亟需可系统发掘功能性致病遗传变异的研究新策略。我们发明了一种整合功能基因组学的策略，可在全基因组范围内系统揭示转录因子结合位点中的功能性致病遗传变异。在本研究中，我们整合分析c-Myc cistromics、千人基因组(1000 Genomes)和TRANSFAC matrix数据，成功注释了12个在肝癌中位于c-Myc cistromics中、并影响c-Myc结合能力的SNPs。通过两阶段、多中心、大样本量病例-对照关联研究，发现上述12个SNPs中，E41 rs122534G>T与肝癌发病风险显著相关。荧光素酶报告基因实验显示，上述SNP位于E41基因启动子区，并可导致E41等位基因差异性表达。EMSA实验结果显示，肝癌风险相关rs122534T等位基因不结合c-Myc。细胞增殖以及裸鼠成瘤实验显示，E41为一种新的肝癌抑癌基因。在“后GWAS时代”，本项目为系统鉴定疾病相关、新的功能性遗传变异和易感基因提供了一种新策略。