HRR通路miRNA及其靶序列SNP与乳腺癌遗传易感性关联分析及功能论证

Breast cancer has become one of the primary malignant tumor of endangering women's health, micro-RNA involved in breast cancer development by regulating a variety of tumor-related genes. Researches indicated micro-RNA target sequences' SNP is an important genetic marker system of tumor, and in recent years a number of GWAS results prompted HRR pathway genes' SNP was related with genetic susceptibility of breast cancer; However, the existing researches about HRR SNP genetic target sequences associated with breast cancer were relatively small and lack of follow-up functional feasibility studies, so they're insufficient to explain this type of SNP's role in the etiology of breast cancer. This research conforms to certain frequency standard HRR micro-RNA seed area target sequences' SNPs by Bio-informatics, through case-control study of large sample, the combined effect of point, multiple points and microRNA-SNP points interaction analysis, on this bases, the regulation and control role of HRR related micrioRNA and its target sequence SNP to breast cancer were explored in by the technologies such as the luciferase reporter system, real-time quantitative PCR, immunohistochemistry, it'll provide a theoretical basis for the high risk assessment, individualization prevention, intervention and treatment of breast cancer.

乳腺癌是危害女性健康的最常见恶性肿瘤之一，micro-RNA通过调控多种肿瘤相关基因参与乳腺癌发生发展。研究表明micro-RNA靶序列SNP是肿瘤一个重要的遗传标记体系，同时近年来多项GWAS结果提示HRR通路基因SNP与乳腺癌的遗传易感性相关；但现有HRR通路基因靶序列SNP与乳腺癌的关联研究规模较小且缺乏后续功能论证，不足以阐述此类SNP在乳腺癌病因中的作用。本研究利用生物信息学选取符合一定频率标准全部HRR相关micro-RNA种子区靶序列SNPs，通过大样本病例对照研究，从单位点、多位点联合作用、microRNA-SNP位点互作分析，并在此基础上通过荧光素酶报告系统、实时定量PCR、免疫组化等技术，探讨HRR相关micrioRNA及其靶序列结合位点SNP对乳腺癌发生的调控作用，为乳腺癌的高发风险估计、个体化预防、干预及治疗提供理论依据。

乳腺癌是危害女性健康的最常见恶性肿瘤之一，micro-RNA通过调控多种肿瘤相关基因参与乳腺癌发生发展。研究表明micro-RNA靶序列SNP是肿瘤一个重要的遗传标记体系，同时近年来多项GWAS结果提示HRR通路基因SNP与乳腺癌的遗传易感性相关；本研究利用生物信息学选取符合一定频率标准全部HRR通路基因相关micro-RNA种子区靶序列SNPs，通过采用生物信息学方法及相关miRNA靶基因预测软件筛选HRR通路miRNA靶序列SNPs；采用病例对照研究方法，在河南汉族人群中选取医院来源的乳腺癌病例450例，按年龄进行频数匹配选择社区来源的健康对照450例，采用PCR-RFLP、CRS-PCR和AS-PCR方法对十二个位点进行基因分型检测，使用SPSS 21.0软件并进行t-检验、卡方检验和非条件logistic回归模型分析，利用Hardy-weinberg平衡在线分析软件判断对照组是否具有人群代表性及SHEsis在线软件进行基因单体型分析，应用MDR 2.0软件分析基因-生殖因素交互作用，且对筛选出的miRNA let-7b进行功能实验验证；研究发现MRE11A rs2155209TC/TC+CC基因型可能增加乳腺癌发病风险；RAD52 rs7963551 C突变等位基因可能降低乳腺癌发病风险；BRCA1 rs8176318 GT/GT+TT可能增加乳腺癌的发病风险；BRCA1 rs15869 AC/ AC+CC显著提高了乳腺癌的发病风险。RAD51基因单体型CA、TA和TG与乳腺癌遗传易感性相关，BRCA1基因单体型GT和TT与乳腺癌遗传易感性相关。MRE11A rs2155209、RAD52 rs7963551、母乳喂养和乳腺癌家族史四因素在乳腺癌发生中具有交互作用；BRCA1 rs8176318、rs12516、怀孕次数和初潮年龄四因素在乳腺癌发生中具有交互作用。pGenesil-1-let-7b过表达载体成功转染到人乳腺癌细胞MCF-7和SKBR3内，并在此两种细胞中成功上调了let-7b的表达水平，let-7b可能负性调节RAD52蛋白的表达。此研究结果为乳腺癌的个体化预防提供理论依据，为深入探讨miRNA对HRR通路靶基因的调控机制提供基线资料。