MiR-17-92基因簇启动子区SNPs及拷贝数变异与结直肠癌发病的关联研究

Colorectal cancer (CRC) is the third most common malignant neoplasm in China, with an increasing incidence in recent years. The etiology, however, is still not fully known. Growing evidence has identified that genetic factors contribute to the development of CRC. Previously, a large number of studies have been performed to investigate the association between single nucleotide polymorphisms (SNPs) and copy number variation (CNV) in encoding genes and susceptibility to CRC. Nevertheless, only a few studies focused on the genetic variants in non-coding genes such as miRNA gene, especially in the promoter region of miRNA gene. It is evident that the level of miR-17-92 cluster was over-expressed in CRC tissues. Several oncogenes or tumor suppressor genes regulate cell cycle, proliferation, and apoptosis by binding to the promoter region of miR-17-92 cluster. Based on this background, we hypothesized that genetic variants in the promoter region of miR-17-92 cluster may be related to the development of CRC. In this study, we plan to examine the relationship between SNPs and CNV in the promoter region of miR-17-92 cluster and risk of CRC by conducting case-control and experimental studies. Additionally, the association between the SNPs and CNV and the high expression of miR-17-92 cluster in CRC will also be detected. We aim to provide evidence to clarify the role of the SNPs and CNV in the promoter region of miR-17-92 cluster playing in the development of CRC.

结直肠癌发病率高居我国恶性肿瘤的第三位，近年来，呈明显上升趋势，然而，其病因尚未完全阐明。多项证据证实，结直肠癌的发生与遗传因素有关。以往大量遗传关联性研究调查了编码基因中的单核苷酸多态性（SNPs）和拷贝数变异（CNV）与肿瘤的易感性，却很少涉及非编码基因如miRNA基因，尤其是发生在miRNA启动子区的遗传变异。研究表明，结直肠癌中miR-17-92基因簇显著高表达，多个癌基因或抑癌基因可通过与miR-17-92基因簇启动子区结合，调控细胞周期、增殖和凋亡等生物学过程，参与肿瘤的形成。由此推测，miR-17-92基因簇启动子区遗传变异可能与结直肠癌的发生有关。本研究拟联合使用SNPs和CNV两个遗传学标记，运用病例对照及功能实验探索miR-17-92基因簇启动子区遗传变异与结直肠癌发病的关联，旨在为阐明miR-17-92基因簇启动子区SNPs和CNV在结直肠癌发生发展中的作用提供依据。

miR-17-92基因簇在结直肠癌中存在差异性表达，然而，原因尚未明晰。本研究收集了874例结直肠癌和1132例无亲缘关系的健康对照样本，运用生物信息学、病例对照和细胞学实验探讨miR-17-92 基因簇启动子区遗传变异与结直肠癌发病的关联。结果显示：rs982873CC基因型显著降低了结肠癌的发病风险（CC vs. TT：校正OR=0.60，95%CI，0.46-0.80；CC vs.TT/TC：校正OR=0.62，95%CI，0.49-0.80）；rs9588884CG和GG基因型显著降低了结直肠癌的发病风险（CG vs. CC：校正OR=0.80，95%CI，0.66-0.98；CG/GG vs. CC: 校正OR=0.72，95%CI，0.59-0.86；GG vs. CC：校正OR=0.46，95%CI，0.35-0.62；GG vs. CC/CG：校正OR=0.53，95%CI，0.40-0.69）。rs1813389在结直肠癌和对照组中分布频率无明显差异。分层分析显示：rs1813389GG基因型在结直肠癌临床Ⅰ-Ⅱ期中的频率明显高于临床Ⅲ-Ⅳ期；GG基因型在无淋巴结转移患者中的频率高于有淋巴结转移者；rs982873TC和TC/CC基因型在结直肠癌临床Ⅰ-Ⅱ期中的频率明显高于临床Ⅲ-Ⅳ期。单倍型分析显示：GCG单倍型在结直肠癌组中的频率明显降低。定量PCR检测miR-17-92在结直肠癌中的表达，发现miR-17和miR-20a在癌组织中高表达，miR-18a低表达。对比多态性分型结果，发现rs982873CC基因型携带者miR-17表达明显降低；rs9588884 GG基因型携带者miR-20a表达明显降低。双荧光素酶报告基因结果显示，rs982873C和rs9588884G等位基因均能显著降低荧光素酶活性。对miR-17-92靶基因进行分析，发现rs9588884GG基因型携带者Bim mRNA表达明显高于CC/CG基因型携带者，提示rs9588884GG基因型降低结直肠癌发病风险的可能原因是降低了miR-20a转录活性和表达，进而增加了抑癌基因Bim的表达。miR-17-92 基因簇启动子区拷贝数变异与结直肠癌无相关性。本项目的完成，不仅鉴定了结直肠癌新的易感基因，而且解释了miR-17-92在结直肠癌中异常表达的原因。