microRNA单核苷酸多态性与新疆哈萨克族人群食管癌发病风险相关性及其机制研究

There was a strong correlation between MicroRNAs (miRNAs) single-nucleotide polymorphisms(SNPs) and tumer etiology and tumer carcinogenesis, To test the hypothesis that SNPs in miRNA related genes may involved in the risk for esophageal cancer, we assessed the associations between esophageal cancer risk and potentially functional single-nucleotide polymorphisms (SNPs) in miRNA-related genes in a case-control study by Mass ARRAY and qRT-PCR technologies. Then to test the expression of related miRNA and their taget genes before and after the Kazakhs esophageal cancer cell will be transfected eukaryotie expression vector for miRNA varients which are likely related to Kazakh Esophageal Cancer Risk.and meanwhile to test the proliferation and Apoptosis of Kazakh′s esophageal cancer cell,analysising affection of related miRNA variants on Kazakh esophageal cancer, elucidating potential mechanisms that miRNA SNPs and miRNA variants involved in process of Kazakh esophageal canceration , studing correlation between miRNA SNPs and etiology and tumer carcinogenesis of Kazakh Esophageal Cancer, eventurally providing new ideas for gene therapy of Kazakh Esophageal Cancer which either early diagnose or prevent based moleculer level.

哈萨克族是我国食管癌高发民族之一；初步研究发现MicroRNAs (miRNAs)单核苷酸多态性与食管癌发病风险相关，本项目以新疆哈萨克族食管癌病例及正常对照人群为研究对象，拟运用Mass ARRAY及qRT-PCR技术在系统检测miRNAs单核苷酸多态性及变异体表达基础上，分析、比较miRNA单核苷酸多态性与新疆哈萨克族食管癌发病风险相关性，并构建与哈萨克族食管癌发病风险相关miRNA变异体真核表达载体，将其转染哈萨克族食管癌细胞，进一步检测细胞转染前后miRNA及其靶基因表达水平的变化和食管癌细胞增殖及凋亡改变，分析miRNA变异体对哈萨克族食管癌发生的影响，初步阐明新疆哈萨克族食管癌中miRNA单核苷酸多态性及变异体在哈族食管癌癌变过程中可能作用机制，探讨miRNA单核苷酸多态性与食管癌病因及发病机制的相关性，为新疆哈萨克族食管癌在分子水平的早期诊断，早期预防和基因治疗提供新的思路。

目的：研究miRNA（miR）单核苷酸多态性（SNPs）与新疆哈萨克族（哈族）食管癌发病风险相关性及在哈族食管癌及哈族健康人群中的基因型分布，并进一步探讨哈族食管癌遗传易感相关miRNA的SNPs不同变异体基因型个体的表达对其成熟miRNA表达的影响，探讨miRNA的SNPs和差异表达的miRNA对哈族食管癌病因及发生发展的影响及可能机制，方法：收集哈族食管鳞癌及健康对照人群样本，运用基质辅助激光解析电离时间飞行质谱（MALDI-TOF MS）技术检测miR34a等miRNA的SNPs位点，卡方检验及Logistic 回归分析miRNA的SNPs位点与哈族食管癌发病风险相关性；qRT-PCR技术检测食管癌发病风险相关miRNA的SNPs位点不同变异体基因型的表达状况及哈族食管癌中无rs72631823多态位点的miR34a的表达；在食管鳞癌细胞系中，运用细胞划痕及侵袭迁移实验检测miR34a对食管癌细胞侵袭、迁移及上皮间叶转化（EMT）的影响，双萤光素酶报告基因方法及Western-blot验证miR34a负向调控的靶基因MMP2/MMP9。结果：获得miRNA的SNPs位点基因型在哈族食管癌及健康人群中的分布，携带miR100（rs1834306T>C）CC基因型个体，miR219-1（rs107822G > A）AA基因型个体和miR124（rs531564C>G）CG基因型个体患食管癌风险明显降低,而携带miR34b（rs11606481C> T）TT基因型个体患食管癌风险显著升高；且miR219-1基因两个SNP位点存在连锁不平衡，携带单体型GT与AT的个体与食管癌发病风险相关；miR219-1（rs107822G > A）位点不同基因型的表达不同；此外，miR34a在食管癌中低表达，且能够抑制食管癌细胞的侵袭迁移和EMT，并验证得到其负向调控的靶基因MMP2/MMP9。结论：miRNA的SNPs位点基因型在哈族食管癌及健康人群中的分布与汉族人群略有不同，且miR100、miR219-1、miR124与miR34b的SNP位点与哈族食管癌发病风险相关，进一步研究发现，食管癌细胞中（EC9706，TE-1）miR34a可能通过负向调控MMP2/MMP9影响食管癌细胞的侵袭、迁移。