全基因组水平microRNA靶基因结合区域遗传变异与结直肠癌易感性及其机制研究

Colorectal cancer (CRC) is one of the common cancers of digestive tract. However, the etiology and mechanisms of CRC is still unknown. CRC is the results of multi-genes and combined with environmental factors, which shows significant individual susceptibility. MicroRNAs (miRNAs) are involved in cell growth, differentiation, and apoptosis, which play a vital role in the development of CRC. It has been known that the expressions of target genes could be regulated by the SNPs in the miRNA target binding region. Here, based on our previous results, we will identify the genome-wide miRNA target binding SNPs via bioinformatics, and investigate the association between these SNPs and CRC risk in a two-stage case-control studies. Furthermore, we will explore the association between the genotypes and clinical phenotypes and the environment-gene interaction. We will determine the potential mechanism of miRNA target binding SNPs on affecting the expression of miRNA and the post-transcriptional regulation of target genes by using a series of molecular biological methods. The results will contribute to clarify the important role of miRNAs in the development of CRC and provide evidence in practice of effective individual prevention, intervention, and therapy of CRC.

结直肠癌是常见的消化道恶性肿瘤之一，其病因和发病机制尚不十分清楚。结直肠癌的发生发展是环境和遗传因素共同作用的结果，且存在明显的个体易感性。微小RNA（microRNA, miRNA）参与了细胞的生长、分化和凋亡等生物学过程，且与结直肠癌的发生发展密切相关，目前已证实miRNA对靶基因的表达调控受到miRNA与其靶基因结合区域的单核苷酸多态性（SNPs）的影响。本课题组在前期研究的基础上，通过生物信息学从全基因组水平确定miRNA与靶基因结合区域的SNPs，拟通过两阶段病例-对照研究，探讨结合区域SNPs与结直肠癌发病风险的关系，分析评价基因型-临床表型关系以及基因-环境交互作用。同时采用一系列细胞分子生物学方法深入探讨SNPs对靶基因转录后翻译调控的影响，最终阐明miRNA结合区域SNPs参与结直肠癌发生发展的可能生物学机制，为今后结直肠癌实施有效的个体化预防、干预和治疗提供理论依据。

目前发现微小RNA（microRNA, miRNA）可通过与靶基因3’-UTR互补结合导致其降解或翻译抑制。本研究基于肿瘤基因图谱（TCGA）、基因表达综合图谱（GEO）及既往文献，筛选出结直肠癌及癌旁组织差异表达的33条miRNAs，并鉴定出1,007个差异表达miRNAs靶基因结合区SNPs。采用两阶段病例-对照研究，分析3’-UTR遗传变异与结直肠癌发病风险及其机制。. 我们在第一阶段病例-对照研究中共纳入1,023例结直肠癌病例及1,306例对照。发现rs1060106、rs631256及rs11245997与罹患结直肠癌风险密切相关（Bonferroni校正后P < 0.05）。随后，我们在855例结直肠癌病例及1,201例健康对照进行第二阶段验证，发现只有位于BET1L基因上的rs11245997（C>T）能够降低结直肠癌的发病风险（第一阶段：调整OR = 0.71，95%CI = 0.61-0.84, P = 5.39E-05；第二阶段：调整OR = 0.82，95%CI = 0.69-0.98, P = 0.029）。通过生物信息学分析及双荧光素酶报告基因实验结果发现，BET1L基因的3’-UTR rs11245997位于miR-140-3p结合区域，其C>T可降低miR-140-3p与靶基因的结合能力，从而导致BET1L的表达上调。另外，BET1L与miR-140-3p的表达在结直肠癌组织中呈负相关（P < 0.01）。进一步的细胞表型实验表明，BET1L作为抑癌基因，异常高表达后，可导致细胞增殖能力降低，凋亡能力减弱，细胞周期在G1期发生阻滞。. 综上所述，BET1L基因3’-UTR的rs11245997遗传变异降低结直肠癌的发病风险，rs11245997可通过改变miR-140-3p与靶基因的结合能力，进而引起BET1L表达水平增高，导致结直肠癌细胞恶性表型的改变，最终参与结直肠癌的发生发展。该研究的发现对结直肠癌的早期诊断及预防具有一定的指导意义。