FOXO基因启动子区 SNP与HBV相关肝癌的关联及其功能研究

Hepatocellular carcinoma(HCC) is one of the most common malignant tumors worldwide with high incidence in South Guangxi in China,which is associated with several different genes and environmental factors.Chronic infection of HBV is one of the important risk factors for HCC,but the carcinogenic mechanisms underlying are unknown.Forkhead box-containing protein O subfamily(FOXO) transcription factor is a pivotal molecule for signaling transduction,whose activity is modulated via multiple mechanisms.It plays an essential role in repair,replication, recombination of DNA,cell cycle and apoptosis.In view of the research in FOXO gene and tumor.It is reasonable to predict that individual difference of SNP of FOXO gene is responsible for widespread HBV infection and HCC associated with region and ethnicity.This project attempts to analyze single nucleotide polymorphism(SNP) of FOXO gene promoter region which may associate with expression and function of gene through bioinformatics.A large case-control study and modern biology technique are conducted,which is used to analyze SNP of FOXO gene associated with the incidence and development of HCC,then identify its biological functions and clarify its influence on FOXO gene and protein expression level in HCC tissues.Based on the research above,this project aims to evaluate the association between HBV-related HCC hereditary susceptibility and SNP of FOXO gene,and the interaction among gene and gene,and the interaction among gene and environment,so as to provide new ideas and a theoretical basis for the mechanism of HCC and comprehensive prevention measures from a new point of view.

肝癌是广西南部高发的恶性肿瘤，是环境和遗传因素综合作用的结果。HBV慢性感染是肝癌的重要危险因素，但其致癌确切机制仍未阐明。FOXO转录因子是一类重要的信号转导分子,它的活性受到多种机制的调节,在DNA修复、复制、重组及细胞周期、细胞凋亡等过程中起重要作用。鉴于FOXO基因与肿瘤等相关性研究成果，有理由推测FOXO基因SNP个体差异是HBV普遍感染，而肝癌发病有地域和种群差异的重要因素。本研究通过生物信息学分析筛选FOXO基因启动子区域可能影响基因表达或基因功能的SNP，运用大样本病例对照分子流行病学研究设计和现代生物学技术等筛选与肝癌发病风险相关的FOXO基因SNP，进一步鉴定其生物学功能，并研究其对肝癌组织FOXO基因及蛋白表达水平的影响，以期探讨SNP、基因表达水平及环境之间的交互作用与HBV相关肝癌发病风险的关系，为从新的角度诠释肝癌发病机理及其综合防治措施提供新的思路和理论依据。

本项目首先实施了以医院为基础的大样本病例对照研究，通过现场流行病学调查方法，共收集了1049名病例与1052名对照的基本人口学与环境危险因素资料，并采集血样提取全基因组DNA。通过生物信息学方法筛选出FOXO1的rs17592236位点、FOXO3的rs4946936位点和FOXO4的rs4503258位点进行研究，采用高通量TaqMan MGB实时荧光定量PCR技术对上述位点进行基因分型，发现rs17592236、 rs4946936和rs4503258位点基因型在病例组和对照组中分布差异均无统计学意义（均有P >0.05）。多因素Logistic回归分析发现，rs17592236位点CT/TT基因型可能降低HCC发病风险[P=0.010，OR（95% CI）=0.699（0.526~0.927）]。 分层分析结果显示rs17592236位点SNP与HCC发病风险存在统计学关联。交互作用分析显示，rs17592236、rs4946936、rs4503258位点多态性与吸烟、饮酒、HBV感染、肝癌家族史4种环境因素均存在交互作用， rs17592236与rs4503258位点SNPs之间存在基因-基因交互作用[P=0.003，OR（95% CI）=0.755（0.628~0.908)]。. 在对上述三个位点的功能预测中发现rs17592236位点是潜在的miRNA has-miR-137结合位点，结合病例对照研究结果，该位点突变可能通过影响FOXO1 3’UTR与miR-137的结合能力从而降低肝癌发病风险。我们分别构建含有野生型与突变型rs17592236的FOXO1 3’UTR序列的miRNA表达载体，培养转染工具细胞293T，将野生型与突变型载体分别与miR-137模拟物共转染293T细胞进行双荧光素酶试验对该位点是否影响miR-137与FOXO1 3’UTR结合进行进一步验证。结果显示rs17592236位点野生型与miR-137结合能力较强，突变型与miR-137结合能力较弱，与预测一致，rs17592236多态性通过调节miR-137结合能力影响HCC发病风险。