miR-143基因启动子区遗传变异影响基因转录调控机制及与宫颈癌发生危险性的研究

Cervical cancer is the second most common diagnosed deadly cancer among females and the most frequently diagnosed malignancy of the female reproductive tract in China. HPV infection is one of the most important factors for cervical cancer, but only a small number of infected people eventually suffer from cervical cancer, indicating that genetic susceptibility involves the reaction of HPV infection. Studies have confirmed that genetic variation (most is SNP) determines individual genetic susceptibility. Recent years, miRNA as a new type of tumor oncogene or suppressor gene has been extensively studied. miRNA involves in a variety of biological pathways, including antiviral immune regulation, therefore, miRNA relevant genetic variation may affect tumor development. Studies have shown that, miR-143 plays as a tumor suppressor gene in the development of cervical cancer. The exact mechanism of its low expression in cervical cancer has not been elucidated. This study will be screening the SNPs within the promoter region of the miR-143 gene which may affect the regulation of gene transcription. The application of cell and molecular techniques, such as reporter assay, EMSA, and real-time quantitative PCR will validated the function of these SNPs. Based on this, we will further analyze the association between the functional SNPs and cervical cancer development, looking for biomarkers for genetic susceptibility. The results of research will have a guiding significance for high risk population screening and individualized prevention of cervical cancer.

宫颈癌高居我国妇女恶性肿瘤前列，患病率居女性生殖道恶性肿瘤的首位。HPV感染是宫颈癌发病的最主要因素之一，但只有少数感染者最终罹患宫颈癌，提示个体对宫颈癌发病具有遗传易感性。研究表明，基因遗传变异（以SNP为主）决定了个体遗传易感性。近年来，miRNA作为一种新的肿瘤癌基因和抑癌基因被广泛研究，参与了包括抗病毒免疫调控在内的多种生物学通路，其遗传变异可影响肿瘤的发生发展。已有研究表明，miR-143发挥抑癌基因作用参与宫颈癌发生发展，其在宫颈癌中低表达的确切机制尚未阐明。本研究将筛选miR-143基因启动子区潜在可影响基因转录调控的SNPs，应用报告基因、EMSA、实时定量PCR等技术和方法对SNPs进行功能学验证；在此基础上，运用分子流行病学研究方法，分析具有调控功能的SNPs与宫颈癌发生危险性的关联性，寻找易感生物标志物。研究结果对宫颈癌高危人群筛检和个体化预防有重要指导意义。

miR-143作为抑癌基因在多肿瘤包括宫颈癌中低表达，其发病机制尚未阐明。本研究通过筛选miR-143基因启动子区潜在可影响基因转录调控的SNPs，采用两阶段的病例-对照研究，分析启动子区遗传变异与宫颈癌遗传易感性的关系，并探讨潜在功能性位点的生物学机制。本研究共筛选出该区域20个SNPs，在第一阶段病例-对照研究中，共纳入571例宫颈癌患者和657对照。结果发现miR-143基因启动子区的rs726953，rs12517403和rs13177623均能够显著增加宫颈癌的发病风险（rs726953: OR =1.71, 95%CI = 1.20-2.43; rs12517403: OR =1.48, 95%CI = 1.15-1.89; rs13177623: OR =1.38, 95%CI = 1.08-1.78）。在第二阶段的954例宫颈癌患者和1339例对照样本中进一步验证，发现rs12517403在共显性模型中仍可增加宫颈癌的发病风险（OR =1.34, 95%CI = 1.11-1.61）。生物信息学预测，发现rs12517403可影响转录因子SP1与miR-143启动子区的结合。通过双荧光素酶报告基因实验，发现相对于rs12517403野生T等位基因，突变C等位基因能够降低miR-143基因启动子区转录活性，从而导致miR-143表达下调。同时，运用生物信息学对TCGA宫颈癌数据库进行分析，发现199例宫颈癌组织携带rs12517403野生纯合基因型TT，73例携带杂合基因型TC，9例携带纯合突变基因型CC，但三者表达值没有显著差异（P=0.281）。rs12517403与组织miR-143表达的关联性需在中国人群宫颈癌组织及癌旁正常组织中进一步验证。综上所述，miR-143基因启动子区rs12517403多态性位点可能通过改变miR-143基因启动子区的转录因子SP1结合能力，从而影响了miR-143的表达，最终导致宫颈癌的遗传易感性改变。该研究的发现对于宫颈癌的早期诊断和预防具有一定的指导意义。