HRR通路miRNA靶基因和LincRNA-ROR SNPs与乳腺癌易感性关联研究及功能分析

发布时间：2018-05-30 20:33

本文选题：乳腺癌 + 易感性　；参考：《郑州大学》2017年硕士论文

【摘要】：乳腺癌是女性最常见的癌症。在中国乳腺癌还是引起中国女性癌症死亡的首要病因。流行病学研究表明乳腺癌的发生发展通常是由遗传、环境、生活方式和生殖危险因素的相互作用引起的。全基因组关联研究(Genome Wide Association Study,GWAS)显示在HRR通路上的DNA修复基因如RAD51、RAD52、XRCC2、BRCA1和BRCA2会导致乳腺癌的发生。在这些基因3’非编码区(3’UTR)的单核苷酸多态性可以通过扰乱现有的或创造miRNAs的结合位点来调控基因的表达水平,进而导致疾病的发生。还有研究表明Linc-ROR在癌症包括乳腺癌的发展中起着重要的作用。然而可以调节这些基因表达水平的潜在功能性单核苷酸多态性(SNP)还未被大量研究。目的本研究的目的是评估十一个SNPs和乳腺癌的遗传易感性之间的相关性以及十一个SNPs与生殖因素之间的交互作用。此外,对于筛选出与乳腺癌有关联性的SNP对应的miRNA和Linc-ROR的标签SNP进行进一步的功能验证,从而探索miRNA调控乳腺癌发生的机制。方法(1)结合NCBI dbSNP数据库、查阅国内外相关文献、预测miRNA结合位点的相关软件以及筛选标签SNP的heploview软件,利用生物信息学方法筛选了HRR通路选定基因(BRCA1、BRCA2、RAD51、RAD52和XRCC2)的miRNA靶序列SNPs(rs8176318、rs12516、rs15869、rs7180135、rs45549040、rs1051669、rs3218550)以及Linc-ROR的标签SNPs(rs1942348、rs4801078、rs6420545和rs9636089);(2)在本次病例对照研究中,根据研究功效(0.9)和最小等位基因频率(0.25),利用PASS软件计算样本量为n=459。本研究纳入了来自河南省的498例乳腺癌患者和按年龄(±2岁)与之进行频数匹配的健康对照者498例。采用限制片段多态性(PCR-RFLP)和等位基因特异性(AS-PCR)的方法对研究对象的DNA样本进行基因分型。分型结果首先进行Hardy-weinberg平衡的检验以保证对照人群的代表性。采用非条件logistic回归模型分析不同基因型与乳腺癌发病风险的关联,并进一步使用多因子降维的方法分析基因与环境的交互作用。此外SHEsis在线软件用作基因BRCA1和Linc-ROR的单体型分析。(3)miRNA-627的过表达载体与miRNA-627抑制剂分别转染到乳腺癌细胞系MDA-MB-231与MCF-7中,利用qRT-PCR的方法来检测不同组内细胞miRNA-627与mRNA BRCA2的相对表达量;采用qRT-PCR的方法检测不同基因型样本的血浆Linc-ROR的表达水平,从而研究Linc-ROR的单核苷酸多态性对自身表达水平的影响。结果(1)BRCA1 rs8176318 GT(OR:1.332;95%CI:1.007-1.761)与GT+TT(OR:1.336;95%CI:1.018-1.753)基因型可以增加乳腺癌的发病风险;此外,BRCA1基因Crs8176318Trs12516的单体型在对照组中的频率明显高于病例组,而BRCA1基因Trs8176318Trs12516的单体型在对照组中的频率显著低于病例组。(2)BRCA2 rs15869 AC(OR:1.524;95%CI:1.141-2.035)基因型也可以增加乳腺癌的发病风险,分层分析显示rs15869 AC+CC基因型在年龄大于50岁(OR:1.486,95%CI:1.047-2.109)、月经初潮年龄≤13岁(OR:1.53,95%CI:1.07-2.188)、绝经年龄50岁(OR:2.185,95%CI:1.094-4.366)、绝经前(OR:1.344,95%CI:1.022-1.767)、妊娠≤2次(OR:1.639,95%CI:1.189-2.261)、流产≤2次(OR:1.392,95%CI:1.1-1.762)、母乳喂养(OR:3.576,95%CI:1.35-9.475)和无前一级亲属肿瘤家族史(OR:1.347,95%CI:1.08-1.679)的人群中增加乳腺癌的发病风险。(3)RAD51/RAD52/XRCC2 RAD51基因rs45549040 AC+CC基因型在妊娠数2次的女性人群中可以降低乳腺癌的发病风险(OR:0.631,95%CI:0.406-0.98);RAD52基因rs1051669 GA+AA基因型可以增加绝经前女性的乳腺癌风险(OR:1.519,95%CI:1.076-2.146);XRCC2 rs3218550 GA+AA基因型在年龄小于50岁(OR:0.627,95%CI:0444-0.885)和月经初潮年龄≤13岁(OR:0.538,95%CI:0.330-0.874)的人群中降低了乳腺癌的风险。(4)Linc-ROR rs4801078 TT(OR:1.791;95%CI:1.195-2.683)可以增加乳腺癌的发病风险;Linc-ROR单体型Trs6420545Crs4801078Trs1942348Trs9636089的频率在对照组相对较高。(5)基因-生殖因素交互作用分析结果:携带rs15869位点C等位基因、初潮年龄≤13岁和怀孕次数≥2次的人群乳腺癌的发病风险是不具有这些危险因素人群的2.39倍(OR:2.39;95%CI:1.04-5.47);rs4801078、怀孕次数以及绝经状态三者之间的交互作用可以提高乳腺的发病风险(OR:2.78;95%CI:2.74-3.61)。(6)实时定量PCR结果:成功转染了pGenesil-1-miRNA-627过表达质粒的乳腺癌细胞系中BRCA2的表达水平较转染了空质粒的对照组显著降低(P0.05),而成功转染miRNA-627抑制物的两个乳腺癌细胞系BRCA2的相对表达水平较其对照组显著增高(P0.05);Linc-ROR的表达水平在rs4801078 CT+TT基因型组的人群中(1.94±0.55)显著高于CC基因型组的人群(1.21±0.49)。结论(1)BRCA1 rs8176318 GT/GT+TT与BRCA2 rs15869 AC基因型可以增加乳腺癌的发病风险;(2)Linc-ROR rs4801078 TT基因型可以增加乳腺癌的发病风险;(3)携带rs15869位点C等位基因、初潮年龄≤13岁和怀孕次数≥2次的人群乳腺癌的发病风险是不具有这些危险因素人群的2.39倍,rs4801078、怀孕次数以及绝经状态三者之间的交互作用可以提高乳腺的发病风险;(4)成功转染的pGenesil-1-miRNA-627过表达质粒在乳腺癌细胞系MCF-7与MDA-MB-231中成功上调了BRCA2的表达水平;rs4801078 CT+TT基因型与健康对照人群中血浆Linc-ROR的表达水平显著相关。
[Abstract]:Breast cancer is the most common cancer in women. In China, breast cancer is the leading cause of cancer death in Chinese women. Epidemiological studies show that the development of breast cancer is usually caused by the interaction of genetic, environmental, lifestyle, and reproductive risk factors. Genome Wide Association Study, GWAS) The DNA repair genes on the HRR pathway such as RAD51, RAD52, XRCC2, BRCA1 and BRCA2 can lead to the occurrence of breast cancer. Single nucleotide polymorphisms in these 3 'non coding regions (3' UTR) can regulate the level of gene expression by disrupting existing or creating miRNAs binding sites, leading to the occurrence of the disease. Linc-ROR plays an important role in the development of cancer, including breast cancer. However, the potential functional single nucleotide polymorphisms (SNP), which can regulate these gene expressions, have not been extensively studied. The purpose of this study was to assess the correlation between the genetic susceptibility to eleven SNPs and breast cancer and eleven SNPs and reproductive causes. In addition, further functional validation of the miRNA and Linc-ROR label SNP corresponding to SNP, which is associated with breast cancer, is further explored to explore the mechanism of miRNA regulation of the occurrence of breast cancer. Method (1) combining the NCBI dbSNP database, consulting the relevant literature at home and abroad, and predicting the related software of the miRNA binding site. And screening the heploview software of the label SNP, using the Bioinformatics Method to screen the miRNA target sequences of the selected HRR pathway (BRCA1, BRCA2, RAD51, RAD52 and XRCC2). (2) In a case-control study, according to the study efficacy (0.9) and the minimum allele frequency (0.25), 498 cases of breast cancer from Henan province and 498 healthy controls who were matched by age (2 years old) were included in the PASS software n=459.. Limited fragment polymorphism (PCR-RFLP) and alleles were used. The specific (AS-PCR) method was used to genotyping the DNA samples of the subjects. The results of the typing were first tested to ensure the representation of the control population. A non conditional logistic regression model was used to analyze the association between the different genotypes and the risk of breast cancer, and the method of multi factor reduction was further used. The interaction between gene and environment. In addition, SHEsis online software is used as a haplotype analysis of gene BRCA1 and Linc-ROR. (3) the overexpression vector of miRNA-627 and miRNA-627 inhibitors are transfected into breast cancer cell line MDA-MB-231 and MCF-7 respectively. The relative expression of miRNA-627 and mRNA BRCA2 in different groups of cells is detected by qRT-PCR method. The qRT-PCR method was used to detect the expression level of plasma Linc-ROR in different genotype samples, and the effect of single nucleotide polymorphism of Linc-ROR on the level of self expression was investigated. Results (1) the genotype of BRCA1 rs8176318 GT (OR:1.332; 95%CI:1.007-1.761) and GT+TT (OR:1.336; 95%CI: 1.018-1.753) could increase the risk of breast cancer. In addition, the frequency of haplotype of BRCA1 gene Crs8176318Trs12516 was significantly higher in the control group than in the case group, while the frequency of the haplotype of BRCA1 gene Trs8176318Trs12516 was significantly lower in the control group than in the case group. (2) the BRCA2 rs15869 AC (OR:1.524; 95%CI:1.141-2.035) genotype also increased the risk of breast cancer, and the stratified analysis showed rs15. 869 AC+CC genotype at age 50 (OR:1.486,95%CI:1.047-2.109), menarche age less than 13 years (OR:1.53,95%CI:1.07-2.188), menopause age 50 (OR:2.185,95%CI:1.094-4.366), Premenopause (OR:1.344,95%CI:1.022-1.767), pregnancy less than 2 times (OR:1.639,95%CI:1.189-2.261), abortion less than 2 times (OR:1.392,95%CI:1.1-1.762), breastfeeding ( OR:3.576,95%CI:1.35-9.475) and the risk of breast cancer (3) the rs45549040 AC+CC genotype of the RAD51/RAD52/XRCC2 RAD51 gene can reduce the risk of breast cancer (OR:0.631,95%CI:0.406-0.98) and RAD52 gene r in a group of women with 2 times of pregnancy. The s1051669 GA+AA genotype can increase the risk of breast cancer in premenopausal women (OR:1.519,95%CI:1.076-2.146); the XRCC2 rs3218550 GA+AA genotype reduces the risk of breast cancer in people younger than 50 years of age (OR:0.627,95%CI:0444-0.885) and menarche age less than 13 years (OR:0.538,95%CI:0.330-0.874). (4) Linc-ROR rs4801078 TT (OR:) (OR:) 1.791; 95%CI:1.195-2.683) can increase the risk of breast cancer; the frequency of Linc-ROR haplotype Trs6420545Crs4801078Trs1942348Trs9636089 is relatively high in the control group. (5) the interaction analysis of gene - reproductive factors: the C allele of the rs15869 locus, the onset of breast cancer in the population with the age of menarche less than 13 years and more than 2 times of pregnancy The risk of the disease was 2.39 times (OR:2.39; 95%CI:1.04-5.47) of those who did not have these risk factors; rs4801078, the number of pregnancies and the interplay between the three of the menopause could improve the risk of breast disease (OR:2.78; 95%CI:2.74-3.61). (6) the real-time quantitative PCR results: the successful transfection of the pGenesil-1-miRNA-627 overexpressed plasmid in the breast cancer The expression level of BRCA2 in the cell lines was significantly lower than that of the control group transfected with empty plasmids (P0.05), while the relative expression level of BRCA2 in two breast cancer cell lines successfully transfected with miRNA-627 inhibitor was significantly higher than that of the control group (P0.05), and the expression level of Linc-ROR was significantly higher than the CC base in the rs4801078 CT+ TT genotype group (1.94 + 0.55). The population of the type group (1.21 + 0.49). Conclusion (1) BRCA1 rs8176318 GT/GT+TT and BRCA2 rs15869 AC genotype can increase the risk of breast cancer; (2) the Linc-ROR rs4801078 TT genotype can increase the risk of breast cancer; (3) carry the C allele of the rs15869 locus, the age of the menarche less than 13 years and the number of pregnancies more than 2 times in the population of breast cancer The risk of the disease is 2.39 times as high as those of the people who do not have these risk factors. Rs4801078, the number of pregnancies and the interaction between the three of the menopause state can increase the risk of breast disease. (4) the successful transfected pGenesil-1-miRNA-627 overexpressed plasmid successfully up-regulated the expression level of BRCA2 in the breast cancer cell line MCF-7 and MDA-MB-231; rs480107 8 the genotype of CT+TT was significantly correlated with the level of plasma Linc-ROR in healthy controls.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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