BER通路基因miRNA结合位点基因多态性与结直肠癌易感性的关联及功能研究

MicroRNA (miRNA) plays a significant role in gene expression through binding to 3'UTR of target genes, and it participates in colorectal cancer occurrence and development. It is suggested that miRNAs mediated abnormal gene expression of base excision repair parthway is associated with colorectal cancer. Since the studies which exploring the association between SNP located in binding site of base excision repair gene and colorectal cancer are usually rare and lack of follow-up function evidences, the real effects of miRNA are expected to be illuminated in colorectal cancer occurrence. In the present study, we intends to use the candidate genes research strategy. Firstly we will select candidate potential functional SNPs located on miRNA binding sites of base excision repair genes (OGG1, XRCC1, UNG, SMUG1, MBD4, TDG and MUTYH etc.) through bioinformatics approach, then we will employ the case-control study design and genetic typing in a huge population to explore the association between the selected SNPs and colorectal cancer susceptibility from unit point to haplotype analysis, finally we will take advantage of molecular biological technique included luciferase reporter gene test and real-time quantitative PCR technology to investigate the real functional roles of these SNPs. The identification of the genetic variation has important significance in screening and preventing the high risk individuals suffering from colorectal cancer.

微小RNA(miRNA)通过与靶基因3’UTR结合对基因转录进行调控，在结直肠癌的发生中起到重要作用。现有研究表明miRNA介导的碱基切除修复(Base excision repair, BER)基因表达异常与结直肠癌发生密切相关，而BER基因3’UTR的单核苷酸多态性变异与结直肠癌的关联研究较少且缺乏后续功能论证。本课题拟采用候选基因为基础的关联研究策略，利用生物信息学方法筛选出BER通路基因（OGG1，XRCC1，UNG，SMUG1，MBD4，TDG和MUTYH等）3’UTR的潜在功能性SNP，采用病例对照研究设计并进行大样本基因分型，从单位点及单体型分析探讨SNP与结直肠癌易感性的关系，并在此基础上通过荧光素酶报告基因实验和实时定量PCR等技术，探讨此类SNP对结直肠癌发生的作用。所识别的遗传变异对于筛查个体罹患结直肠癌的风险和人群预防有重要意义。

结直肠癌是一种严重危害人类健康的常见恶性肿瘤。开展结直肠癌的遗传易感性研究，明确影响结直肠癌易感性的遗传位点及其作用机制，对于高危人群的筛检和结直肠癌的预防有重要意义。miRNA介导的碱基切除修复(Base excision repair, BER)基因3’UTR的单核苷酸多态性变异与结直肠癌的关联研究较少且缺乏后续功能论证，因此探讨BER基因miRNA结合位点SNP对结直肠癌发生的作用，对于早期预警个体罹患结直肠癌的风险有重要意义。本课题采取候选基为基础的研究策略，选取BER通路中的关键基因作为靶基因，采用生物信息学方法确定靶点SNP，通过大样本病例对照研究分析其与结直肠癌易感性的关联。对鉴定得到的阳性SNP利用细胞生物学方法分析其与靶miRNA的相互作用及作用机制，最终阐明此类SNP与结直肠癌易感性之间的关系。本研究发现：mir-27a rs895819 SNP位点GG基因型及G等位基因是结直肠癌发病的危险因素（显性模型: OR = 1.15, 95% CI: 1.02–1.29, p = 0.02；隐性模型: OR = 1.49, 95% CI: 1.27–1.76, p < 0.001；纯合子模型: OR = 1.53, 95% CI: 1.28–1.83, p < 0.001）；等位基因模型: OR = 1.21, 95% CI: 1.11–1.31, p < 0.001）；结直肠癌中异常表达的miRNA主要集中于mir-17-92基因簇，但课题组分析该基因簇对结直肠癌预后影响未发现直接联系（aHR=1.49, 95%CI:0.98-2.27）。同期细胞学实验发现与正常结直肠黏膜细胞相比，mir-19在结直肠癌细胞系中呈现高表达状态，提示mir-19在结直肠癌中可能发挥癌基因作用。采用脂质体转染方法进行mir-19过表达和干扰实验，发现过表达mir-19可抑制HCT116细胞凋亡，Wnt/β-catenin信号通路可能是其发挥作用的主要通路。本研究共计公开发表SCI论文5篇，培养硕士研究生4名。已完成项目计划书中发表SCI论文2-3篇，培养研究生3-4名的目标。