基于前期GWAS的肺癌发生相关lncRNA基因SNP的筛选、验证及作用机制研究

Long non-coding RNAs (lncRNAs) may play important roles in the development of cancer, however its key points are not clear. This project will identify lncRNAs and their single nucleotide polymorphisms (SNPs) based on previous genome wide association study (GWAS) results of lung cancer using systematic review and bioinformatic methods. The case-control study including large sample size population will be applied to study the relationship between lncRNA SNPs and lung cancer risks, as well as gene-gene and gene-environment interaction. For significantly associated SNPs, biological significance will be explored. The impact of SNPs on the expression, structure and combination with microRNAs of lncRNA will be explored by real-time PCR, dual luciferase reporter gene assay system and bioinformatic methods. The influence of lncRNAs on proliferation, apoptosis, cell cycle and colony formation of lung cancer cell will be studied. By using the lncRNA knockdown and overexpression, combined with RNA pull-down, immunoprecipitation, real-time PCR, Western Blot, siRNA interference, the study will demonstrate that lncRNAs interact with proteins and regulate signal pathways. This project aims to find new lncRNAs and their SNPs associated with the susceptibility of lung cancer as well as their key roles in the development of cancer, which are valuable in understanding the molecular mechanism of lung cancer development.

长链非编码RNA (lncRNA)可能在肿瘤发生中起重要作用，但关键环节尚未阐明。本研究基于前期肺癌全基因组关联研究（GWAS）结果，拟利用生物信息学等方法筛选lncRNA基因单核苷酸多态性（SNP）；然后采用大样本人群病例对照研究方法探讨这些SNP对肺癌发生的危险度，得到与肺癌相关的SNP，在此基础上探讨相关的基因-基因和基因-环境在肺癌发生中的交互作用；最后对已验证确定的lncRNA及其SNP进行相关的生物学功能研究，探讨这些SNP对所在lncRNA折叠结构、表达及与微小RNA结合能力的影响，评价lncRNA对肺癌细胞系增殖、凋亡、细胞周期及细胞集落形成等恶性表型的影响，寻找并验证lncRNA特异性结合蛋白及调控的信号通路。旨在发现新的与肺癌发生相关的lncRNA及其SNP，揭示它们在致癌过程中的关键作用，研究结果对深入阐释肺癌发生发展的内在机制和遗传易感性有重要学术价值。

长链非编码RNA(lncRNA)在肺癌发生中起重要作用，但关键环节尚未阐明。本课题利用生物信息学和系统评价等方法，筛选出44个位于lncRNA基因区域的单核苷酸多态性（SNP）；通过分子流行病学方法探讨这些SNP对肺癌发生的影响，结果发现lncRNA中HOTAIR的rs4759314和rs12826786、HOXA11-AS的rs17427875、CARD8-AS1的rs7248320、MEG3的rs4081134、CDKN2B-AS1的rs2151280、PRNCR1的rs13252298和rs1456315、TERC的rs2293607、HULC的rs1041279、ANRIL的rs2151280等遗传变异与肺癌发生的关联有统计学意义。基因-环境交互作用分析未发现这些SNP与环境危险因素存在相加或相乘交互作用。本研究发现HOXA11-As和CCAT1等lncRNA在肺癌组织中的表达明显高于正常组织。生物学功能研究表明：1. HOTAIR可通过与miR-149-5p结合来抑制miR-149-5p与HNRNPA1的mRNA结合，从而提高HNRNPA1的mRNA表达量并提高HNRNPA1蛋白的表达；过表达的HOTAIR可以促进肺癌细胞的迁移、侵袭等能力，并且可以提高细胞的增殖能力，使细胞处于更高的分裂活跃期；双荧光素酶报告基因检测结果显示miR-149-5p与HOTAIR靶向结合，并且miR-149-5p与HNRNPA1也有靶向结合的关系；2. HOXA11-AS的rs17427875可影响该lncRNA的表达水平，T等位基因显著增加HOXA11-AS的表达量，降低其对应编码基因HOXA11的蛋白质表达水平，从而影响肺腺癌细胞的增殖、迁移和凋亡等生物学进程；回复实验结果证明HOXA11-AS可作为原癌基因调控肺腺癌细胞的增殖迁移。因此，本课题得出结论：lncRNA基因区域的SNP与肺癌的易感性有关，SNP可通过影响lncRNA的表达水平及竞争性内源RNA的机制从而影响肺癌的发生。本研究提示lncRNA基因SNP可作为肺癌发生发展重要的标志物，这不仅为揭示lncRNA在致肺癌过程中的作用、阐明lncRNA对基因表达的调控提供一定的理论依据，而且对深入阐释肺癌发生发展的内在机制和遗传易感性有重要学术价值。