LncRNAs调控型遗传变异与中国人群肝癌易感性及机制研究

The abnormal expression of lncRNAs is an important mechanism in promoting the malignant transformation of hepatocellular carcinoma. It is particular importance in understanding the biological mechanism of hepatocarcinogenesis to explore the regulation mechanisms of lncRNAs. Emerging biological evidence indicated that both proximal and distal regulatory elements play important roles in transcriptional regulation. Genetic variants in the regulatory elements may involve in gene regulation by altering the activity of the proximal promoter or distal enhancer and the chromatin looping interaction. Therefore, the regulatory variants at the hepatocellular carcinoma predisposition loci may play critical roles in cancer predisposition by modulating lncRNAs expression. The study may comprehensively integrate 3D chromatin structures Hi-C data, the Cancer Genome Atlas (TCGA) database and ChIP-Seq data of histone marks and transcription factors to prioritize genetic variants by the potential functionality on modulating lncRNAs at the hepatocellular carcinoma predisposition loci. Here a two-staged case-control study with large samples will be conducted to identify the association between lncRNAs regulatory variants and hepatocellular carcinoma risk. The biological mechanisms will be further assessed by functional analyses to clarify how to modulate of lncRNAs by risk-associated SNPs and illuminate mechanisms of hepatocellular carcinoma predisposition. This will advance in our understanding of the identification of causative variants predisposing to hepatocellular carcinoma. Furthermore, the identified causative variants revealed by this study can be used as genetic markers for high-risk population screening and personalized strategies to prevent hepatocellular carcinoma.

LncRNAs异常表达是导致肝癌细胞恶性转化的重要分子机制，揭示其表达调控机制对解析肝癌病因尤为重要。生物学证据表明，基因转录调控受到近端和远端调控元件的共同作用，调控元件中的遗传变异很可能通过影响近端启动子或者远端增强子的活性，甚至影响三维空间折叠，来调控基因的表达。因此，肝癌易感区域内遗传变异很可能通过调控lncRNAs表达而影响肝癌发生。本研究系统整合三维基因组Hi-C数据、癌症基因组图集TCGA数据、以及组蛋白修饰和转录因子结合的ChIP-Seq数据，在肝癌易感区域内筛选lncRNAs调控型遗传变异；然后通过两阶段病例-对照研究，揭示候选遗传变异与肝癌易感性的关系；最后结合生物学实验解析遗传变异调控lncRNAs表达以及lncRNAs异常表达后促进肝癌的分子机制。本研究不仅解释了遗传变异引起肝癌易感的生物学机制，所识别的遗传变异也可能为肝癌高危人群筛检和个体化防治提供线索和依据。

本研究结合生物信息学、流行病学和分子生物学等多种技术手段，系统阐述LncRNAs调控型遗传变异在中国人群肝癌发生发展中的作用。本研究发现易感区域内LINC01149 rs2844512 C等位基因显著增加肝癌发病风险(OR=1.21, 95% CI=1.11-1.32)。通过双荧光素酶报告基因实验和表达定量性状遗传位点分析发现LINC01149 [C]能够与miR-128-3p结合，进而增加MICA的表达水平。体内和体外实验表明，LINC01149[C]稳转肝癌细胞培养液中可溶性MICA蛋白增加，同时携带LINC01149 CC基因型的肝癌患者血清可溶性MICA显著增加，从而增强了肝癌细胞的免疫逃避作用。此外，我们还发现影响增强子活性的LncRNA调控型遗传变异rs2647046 A等位基因显著增加肝癌发病风险(OR = 1.26, 95%CI = 1.11-1.43)。通过双荧光素酶报告基因实验、凝胶电泳迁移率变动实验、超迁EMSA实验和染色质构象捕获技术3C，发现调控型遗传变异rs2647046对LncRNA HLA-DQB1-AS1有远端调控作用，且细胞增殖实验显示LncRNA HLA-DQB1-AS1高表达显著增加肝癌细胞系增殖能力。所发掘的遗传变异有可能作为标志物，用于肝癌高风险个体的筛查及肝癌个体化防治。