补体通路调节基因遗传变异与肝细胞癌易感性及其机制研究

Hepatocellular carcinoma (HCC) is the second most frequent cause of cancer death in our country; however,the pathogenesis of HCC has not been fully clarified.A number of evidence reveals that the occurrence and development of HCC were associated with genetic factor.The complement system plays an important role in the occurrence and development of cancer. The recent studies indicated that C3a and C5a issuing from the complement activation could promote cancer growth by different ways and different mechanisms. However, the activation of complement system was controlled by a variety of complement regulating proteins. Therefore, the single nucletide polymorphism (SNP) of the complement regulating gene which encoded the complement regulating proteins could influence the cancer risk of different individuals. In recent years,there is a few studies about the polymorphism of the complement regulating gene in the complement pathway and cancer susceptibility. However, the relationship between the polymorphism and HCC is still unclear. The environmental exposure data (such as smoking, drinking, etc.) in HCC group and healthy control group were surveyed and collected by epidemiological methods in this case-control study. The high-throughput SNPs detection technology was used to screen tag SNPs associated with HCC. In order to identify the risk loci which were associated with HCC, our research team will estimate the effect of tagSNPs, gene-gene, gene-environment on HCC risk based on our previous work. Then, the luciferase reporter gene, real time-PCR and western blotting methods would be used to detect the expression of the gene in which the tagSNPs distribution was significantly different in the HCC and controls groups, which would attempt to clarify the pathogenesis of HCC by the SNPs of complement regulating gene in the complement pathway. The identified SNPs, which were associated with HCC, might be used as potential genetic markers for HCC screening in high-risk individuals and could provide important reference for individualized intervention and prevention of HCC.

肝癌高居我国癌症死亡的第二位，然而其发病机制尚未阐明。近年的研究揭示补体系统激活后产生的C3a及C5a可通过不同途径或机制促进肿瘤生长。然而，补体激活受多种补体调节蛋白调节，那么编码补体调节蛋白的基因遗传变异可能影响个体对肿瘤的发病风险。目前国内外这方面的研究较少，与肝癌的关系仍不清楚。本课题通过病例-对照研究设计，流行病学调查并收集肝癌患者和健康对照者的吸烟、饮酒等环境暴露资料；运用SNPs高通量检测技术筛选补体调节基因中与肝癌发病相关SNPs。课题组在该基础上对补体通路调节基因的SNPs、基因－基因、基因－环境与肝癌进行关联分析，找出与肝癌相关的风险位点；并通过荧光素酶报告基因、RT-PCR和免疫印迹等研究这些风险位点多态性对基因表达变化的影响，尝试阐明补体通路调节基因遗传变异对肝癌发病的影响机理。研究发现的阳性遗传变异可为我国肝癌高危人群的筛查以及肝癌的个体化预防和干预提供重要参考。

本研究联合分子流行病学，分子生物学，生物信息学等方法，在四川地区肝癌-对照组中（肝癌1200例，对照1250例）阐述补体调节基因遗传变异与肝癌易感性的关联及其机制。研究共纳入补体系统9个基因中42个标签SNPs，研究分为发现组（2007-2014年）及验证组（2017-2018年）。研究结果如下：1. 发现组初筛揭示CD46 rs2796267 及 rs2796268 均为肝癌易感性的阳性位点，但经验证组人群验证后发现仅 rs2796267 与肝癌易感性相关。与野生基因型AA相比，突变纯合子GG明显增加肝癌发病风险(OR=2.03，95% CI= 1.34-3.08)。2. 分层分析发现，CD46 rs2796267增加男性及年龄小于65岁者的肝癌风险，基因-环境因素分析发现CD46 rs2796267 AG/GG基因型显著增加饮酒(OR=2.97，95% CI= 1.76-4.99)及吸烟者(OR=2.84，95% CI= 1.74-4.64)的肝癌风险，同时其明显增加HBsAg阳性者罹患肝癌的风险(OR=2.16，95% CI= 1.45-3.21)。3. 据肝癌临床病理类型分层发现，CD46 rs2796267可增加不同临床病理类型肝癌的风险。4. 功能研究方面，双荧光素酶报告基因实验及凝胶迁移实验揭示：rs2796267 突变等位基因(G)较野生等位基因(A)增加了该位点与转录因子STAT 5a 的结合能力，从而增强了CD46基因表达导致荧光表达水平的增加。后续mRNA表达方面，RT-PCR结果显示GG基因型较AA基因型的表达量明显增加。免疫组化及Western-bolt结果进一步证实了RT-PCR的结果，即GG基因型蛋白表达量较AA基因型高。综上揭示CD46 基因启动子区rs2796267 A/G变异可能通过增加与转录因子STAT 5a 的结合从而增强了CD46基因的转录及翻译导致CD46表达增加，进而增加了肝癌的易感性。5. 本研究还发现CD35 rs7525160 为肝癌的阳性关联位点；但是该结果仅在验证群组中有意义，故该结果有待增加样本量证实。综上，本研究所发现的CD46 rs2796267 位点可作为肝癌易感人群筛查的遗传标志物之一，rs2796267与饮酒、吸烟及HBsAg的基因-环境相互作用可为肝癌三级预防措施的制定提供科学基础。