HRR通路miRNA靶基因SNP与乳腺癌易感性关联研究及功能分析
发布时间：2019-07-01 18:29
【摘要】：乳腺癌是女性最常见的恶性肿瘤之一,近年来其发病率呈不断上升的趋势,且发病年龄日益趋于年轻化,严重影响了女性的身心健康。目前研究认为乳腺癌的发生发展主要是女性生殖因素和遗传因素共同作用的结果,多项全基因组关联研究(Genome Wide Association Study,GWAS)发现同源重组修复(Homology Recombination Repair,HRR)通路基因单核苷酸多态性(SNP)是个体DNA损伤修复能力差异的分子遗传学基础,mi RNAs种子区以碱基互补配对方式与HRR通路靶基因m RNA 3’非翻译区(3’UTR)特异结合,导致m RNA降解或抑制其翻译从而影响HRR通路基因的表达和功能,进而DNA损伤修复能力下降或缺失等引发的基因组不稳定是乳腺癌发生的重要原因。目的探讨HRR通路mi RNA靶基因单核苷酸多态性(MRE11A rs2155209、NBS1rs2735383、RAD51 rs963917和rs963918及RAD52 rs7963551)与乳腺癌遗传易感性的关联以及与女性生殖因素的交互作用,为乳腺癌的个体化预防提供理论依据,且对筛选出的mi RNA let-7b进行功能实验验证,为mi RNA对HRR通路靶基因的调控机制提供依据。方法(1)结合国内外文献数据库,采用生物信息学方法及相关mi RNA靶基因预测软件筛选HRR通路mi RNA靶序列SNPs;(2)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和AS-PCR方法对五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RAD51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;(3)采用分子克隆技术构建let-7b过表达载体,结合let-7b抑制剂分别转染到乳腺癌细胞系MCF-7和SKBR3内,采用q RT-PCR分析let-7b和m RNA RAD52的相对表达量及Western-blot分析RAD52蛋白表达水平进而判断RAD52是否是let-7b的靶基因。结果(1)单位点分析结果显示:MRE11A rs2155209 TC(OR调整=1.87,95%CI=1.23-2.86)和TC+CC(OR调整=1.86,95%CI=1.23-2.80)基因型可增加乳腺癌发病风险,RAD52 rs7963551 AC(OR调整=0.67,95%CI=0.48-0.87)、CC(OR调整=0.36,95%CI=0.24-0.58)和AC+CC(OR调整=0.71,95%CI=0.59-0.82)基因型可降低乳腺癌发病风险;(2)分层分析结果显示:在具有乳腺癌家族史的女性中,rs2155209 TC+CC基因型(OR=1.49,95%CI=1.06-2.08)和rs963917 TC+CC基因型(OR=1.78,95%CI=1.04-3.05)显著增加乳腺癌发病风险,rs2735383 GC+GG基因型在未绝经女性中可显著降低乳腺癌发病风险(OR=0.47,95%CI=0.33-0.66),rs963918AG+GG基因型在生育次数≤2次的女性中乳腺癌发病风险较高(OR=1.64,95%CI=1.10-2.45),rs7963551 AG+GG基因型在母乳喂养女性中的乳腺癌发病风险较高(OR=1.49,95%CI=1.13-1.96),在PR阳性乳腺癌患者中,rs7963551 AC+CC(OR=1.82,95%CI=1.33-2.48)基因型均显示与较高的乳腺癌发病风险相关;(3)五个位点联合效应分析结果显示:随着突变位点个数的增加,乳腺癌的发病风险呈现增加的趋势(Ptrend=0.003);(4)单体型分析结果显示:RAD51基因Trs963917Ars963918和Trs963917Grs963918单体型可降低乳腺癌发病风险(OR调整=0.53,95%CI=0.4-0.68),而Trs963917Ars963918和Trs963917Grs963918单体型与乳腺癌发病风险增加相关(OR调整=1.28,95%CI=1.05-1.57和OR调整=1.31,95%CI=1.09-1.62);(5)基因-生殖因素交互作用显示:携带rs2155209位点C等位基因和rs7963551位点A等位基因及具有乳腺癌家族史和母乳喂养经历的“高危”女性乳腺癌发病风险是非上述组合的“低危”女性的3.59倍;(6)单双酶切电泳及测序结果显示:p Genesil-1-let-7b过表达载体构建成功;q RT-PCR结果显示在MCF-7细胞和SKBR3细胞中,let-7b和m RNA RAD52在let-7b过表达组和抑制剂组中的相对表达量差异均具有统计学意义(P0.05);(7)Western-blot结果显示:在MCF-7细胞中,let-7b过表达组中RAD52蛋白表达水平下调,let-7b抑制剂组中RAD52蛋白表达水平上调;在SKBR3细胞中,RAD52蛋白在let-7b过表达组和抑制剂组中的表达量差异均无统计学意义(P0.05)。结论(1)MRE11A rs2155209 TC/TC+CC基因型可能增加乳腺癌发病风险,RAD52rs7963551 C突变等位基因可能降低乳腺癌发病风险,RAD51基因单体型CA、TA和TG与乳腺癌遗传易感性相关,rs2155209、rs7963551、母乳喂养和乳腺癌家族史在乳腺癌发生中具有交互作用;(2)p Genesil-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,在MCF-7细胞中RAD52可能是let-7b的靶基因,且let-7b负性调节RAD52蛋白的表达。
[Abstract]:Breast cancer is one of the most common malignant tumors in women. In recent years, the incidence of breast cancer has been increasing, and the onset age is becoming more and more younger, which seriously affects the physical and mental health of women. At present, the development of breast cancer is considered to be the result of the common effects of female reproductive and genetic factors, and the Genome Wide Association Study (GWAS) has found homologous recombination repair (HAs). The HRR pathway gene, a single nucleotide polymorphism (SNP), is a molecular genetic basis for individual DNA damage repair, and the mi RNAs seed region is specifically combined with the HRR pathway target gene m RNA 3 'non-translated region (3' UTR) in a base complementary pairing manner, It is an important cause of the occurrence of breast cancer due to the degradation of m-RNA or the inhibition of its translation so as to influence the expression and function of the HRR pathway gene. Objective To study the association between the single-nuclear polynucleotide polymorphism (MRE11A rs2155209, NBS1rs2735383, RAD51 rs963917 and rs963918 and RAD52 rs7963551) of the HRR-channel mi-RNA target gene and the genetic susceptibility of breast cancer and the interaction with female reproductive factors, to provide a theoretical basis for the individual prevention of breast cancer. The function experiment of the screened mi-RNA let-7b was carried out to provide the basis for the control mechanism of the target gene of the HRR via the mi-RNA. Methods (1) The method of bioinformatics and relevant mi-RNA target gene prediction software was used to screen HRR-channel mi-RNA target sequence SNPs; (2) case-control study was used to select 450 cases of breast cancer from the Han population of Henan. Using the method of PCR-RFLP and AS-PCR, we used the SPSS 21.0 software to carry out t-test, chi-square test and non-conditional logistic regression model. using the Hardy-weinberg equilibrium on-line analysis software to judge whether the control group has the population representative and the SHEsis online software for carrying out the RAD51 gene haplotype analysis, and using the MDR 2.0 software to analyze the interaction of the gene-reproduction factors; and (3) constructing the let-7b over-expression vector by using the molecular cloning technology, The relative expression of the let-7b and m-RNA RAD52 was analyzed by q-RT-PCR and the expression level of the RAD52 protein was analyzed by Western-blot to determine whether the RAD52 was the target gene of the let-7b. Results (1) The results of unit point analysis showed that the genotype of MRE11A rs2155209TC (OR adjustment = 1.87,95% CI = 1.23-2.86) and TC + CC (OR adjustment = 1.86,95% CI = 1.23-2.80) could increase the risk of breast cancer, RAD52 rs7963551 AC (OR adjustment = 0.67,95% CI = 0.48-0.87), CC (OR adjustment = 0.36,95% CI = 0.24-0.58) and AC + CC (OR adjustment = 0.71,95% CI = 0.59-0.82) genotype could reduce the risk of breast cancer. (2) The results of stratified analysis showed that rs2155209TC + CC genotype (OR = 1.49,95% CI = 1.06-2.08) and rs963917TC + CC genotype (OR = 1.78,95% CI = 1.04-3.05) significantly increased the risk of breast cancer in women with family history of breast cancer. The risk of breast cancer (OR = 0.47,95% CI = 0.33-0.66), rs963918AG + GG genotype in breast-fed women was higher (OR = 1.64,95% CI = 1.10-2.45) and rs7963551AG + GG genotype in breast-fed women with high risk of breast cancer (OR = 1.49,95% CI = 1.13-1.96), and in PR-positive breast cancer patients, the risk of breast cancer in breast-fed women was higher (OR = 1.64,95% CI = 1.10-2.45), and rs7963551AG + GG genotype was higher in breast-fed women (OR = 1.49,95% CI = 1.13-1.96). The genotype of rs7963551 AC + CC (OR = 1.82,95% CI = 1.33-2.48) was related to the risk of breast cancer. (3) The results of the combined effects of five sites showed that with the increase of the number of mutation sites, the risk of breast cancer was increased (Ptrend = 0.003); and (4) the results of the haplotype analysis showed that: The single type of the RAD51 gene, Trs963917Ars963918 and Trs963917Grs963918, can reduce the risk of breast cancer (OR adjustment = 0.53,95% CI = 0.4-0.68), while Trs963917Ars963918 and Trs963917Grs963918 are associated with increased risk of breast cancer (OR adjustment = 1.28,95% CI = 1.05-1.57 and OR adjustment = 1.31,95% CI = 1.09-1.62); and (5) gene-reproduction factor interaction display: The risk of female breast cancer, which carried the locus A of rs2155209 and the locus A of rs7963551 and the family history of breast cancer and the high risk of breast-feeding, was 3.59 times that of the low-risk female with the combination of the above-mentioned combination; (6) the results of single-enzyme digestion and sequencing showed that: The expression vector was successfully constructed by p-Genesil-1-let-7b. The results of q-RT-PCR showed that in the MCF-7 cells and SKBR3 cells, the relative expression levels of the let-7b and m-RNA RAD52 in the let-7b overexpression group and the inhibitor group were statistically significant (P0.05); and (7) Western-blot results showed that in the MCF-7 cells, The expression of RAD52 in the let-7b overexpression group was down-regulated, and the expression level of RAD52 in the let-7b inhibitor group was up-regulated; in the SKBR3 cells, the expression of the RAD52 protein in the let-7b overexpression group and the inhibitor group was not statistically significant (P0.05). Conclusion (1) The genotype of the MRE11A rs2155209TC/ TC + CC may increase the risk of breast cancer, and the RAD52rs7963551C mutant allele may reduce the risk of breast cancer, and the RAD51 gene Monomer CA, TA and TG are related to the genetic susceptibility of breast cancer, rs2155209, rs7963551, The family history of breast-feeding and breast cancer has an interaction in the occurrence of breast cancer; (2) the expression vector of the p-Genesil-1-let-7b is successfully transfected into the human breast cancer cells MCF-7 and SKBR3, and the expression level of the let-7b is successfully raised in the two cells, and the RAD52 in the MCF-7 cell may be the target gene of the let-7b, And let-7b negatively regulate the expression of the RAD52 protein.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9
本文编号：2508705
[Abstract]:Breast cancer is one of the most common malignant tumors in women. In recent years, the incidence of breast cancer has been increasing, and the onset age is becoming more and more younger, which seriously affects the physical and mental health of women. At present, the development of breast cancer is considered to be the result of the common effects of female reproductive and genetic factors, and the Genome Wide Association Study (GWAS) has found homologous recombination repair (HAs). The HRR pathway gene, a single nucleotide polymorphism (SNP), is a molecular genetic basis for individual DNA damage repair, and the mi RNAs seed region is specifically combined with the HRR pathway target gene m RNA 3 'non-translated region (3' UTR) in a base complementary pairing manner, It is an important cause of the occurrence of breast cancer due to the degradation of m-RNA or the inhibition of its translation so as to influence the expression and function of the HRR pathway gene. Objective To study the association between the single-nuclear polynucleotide polymorphism (MRE11A rs2155209, NBS1rs2735383, RAD51 rs963917 and rs963918 and RAD52 rs7963551) of the HRR-channel mi-RNA target gene and the genetic susceptibility of breast cancer and the interaction with female reproductive factors, to provide a theoretical basis for the individual prevention of breast cancer. The function experiment of the screened mi-RNA let-7b was carried out to provide the basis for the control mechanism of the target gene of the HRR via the mi-RNA. Methods (1) The method of bioinformatics and relevant mi-RNA target gene prediction software was used to screen HRR-channel mi-RNA target sequence SNPs; (2) case-control study was used to select 450 cases of breast cancer from the Han population of Henan. Using the method of PCR-RFLP and AS-PCR, we used the SPSS 21.0 software to carry out t-test, chi-square test and non-conditional logistic regression model. using the Hardy-weinberg equilibrium on-line analysis software to judge whether the control group has the population representative and the SHEsis online software for carrying out the RAD51 gene haplotype analysis, and using the MDR 2.0 software to analyze the interaction of the gene-reproduction factors; and (3) constructing the let-7b over-expression vector by using the molecular cloning technology, The relative expression of the let-7b and m-RNA RAD52 was analyzed by q-RT-PCR and the expression level of the RAD52 protein was analyzed by Western-blot to determine whether the RAD52 was the target gene of the let-7b. Results (1) The results of unit point analysis showed that the genotype of MRE11A rs2155209TC (OR adjustment = 1.87,95% CI = 1.23-2.86) and TC + CC (OR adjustment = 1.86,95% CI = 1.23-2.80) could increase the risk of breast cancer, RAD52 rs7963551 AC (OR adjustment = 0.67,95% CI = 0.48-0.87), CC (OR adjustment = 0.36,95% CI = 0.24-0.58) and AC + CC (OR adjustment = 0.71,95% CI = 0.59-0.82) genotype could reduce the risk of breast cancer. (2) The results of stratified analysis showed that rs2155209TC + CC genotype (OR = 1.49,95% CI = 1.06-2.08) and rs963917TC + CC genotype (OR = 1.78,95% CI = 1.04-3.05) significantly increased the risk of breast cancer in women with family history of breast cancer. The risk of breast cancer (OR = 0.47,95% CI = 0.33-0.66), rs963918AG + GG genotype in breast-fed women was higher (OR = 1.64,95% CI = 1.10-2.45) and rs7963551AG + GG genotype in breast-fed women with high risk of breast cancer (OR = 1.49,95% CI = 1.13-1.96), and in PR-positive breast cancer patients, the risk of breast cancer in breast-fed women was higher (OR = 1.64,95% CI = 1.10-2.45), and rs7963551AG + GG genotype was higher in breast-fed women (OR = 1.49,95% CI = 1.13-1.96). The genotype of rs7963551 AC + CC (OR = 1.82,95% CI = 1.33-2.48) was related to the risk of breast cancer. (3) The results of the combined effects of five sites showed that with the increase of the number of mutation sites, the risk of breast cancer was increased (Ptrend = 0.003); and (4) the results of the haplotype analysis showed that: The single type of the RAD51 gene, Trs963917Ars963918 and Trs963917Grs963918, can reduce the risk of breast cancer (OR adjustment = 0.53,95% CI = 0.4-0.68), while Trs963917Ars963918 and Trs963917Grs963918 are associated with increased risk of breast cancer (OR adjustment = 1.28,95% CI = 1.05-1.57 and OR adjustment = 1.31,95% CI = 1.09-1.62); and (5) gene-reproduction factor interaction display: The risk of female breast cancer, which carried the locus A of rs2155209 and the locus A of rs7963551 and the family history of breast cancer and the high risk of breast-feeding, was 3.59 times that of the low-risk female with the combination of the above-mentioned combination; (6) the results of single-enzyme digestion and sequencing showed that: The expression vector was successfully constructed by p-Genesil-1-let-7b. The results of q-RT-PCR showed that in the MCF-7 cells and SKBR3 cells, the relative expression levels of the let-7b and m-RNA RAD52 in the let-7b overexpression group and the inhibitor group were statistically significant (P0.05); and (7) Western-blot results showed that in the MCF-7 cells, The expression of RAD52 in the let-7b overexpression group was down-regulated, and the expression level of RAD52 in the let-7b inhibitor group was up-regulated; in the SKBR3 cells, the expression of the RAD52 protein in the let-7b overexpression group and the inhibitor group was not statistically significant (P0.05). Conclusion (1) The genotype of the MRE11A rs2155209TC/ TC + CC may increase the risk of breast cancer, and the RAD52rs7963551C mutant allele may reduce the risk of breast cancer, and the RAD51 gene Monomer CA, TA and TG are related to the genetic susceptibility of breast cancer, rs2155209, rs7963551, The family history of breast-feeding and breast cancer has an interaction in the occurrence of breast cancer; (2) the expression vector of the p-Genesil-1-let-7b is successfully transfected into the human breast cancer cells MCF-7 and SKBR3, and the expression level of the let-7b is successfully raised in the two cells, and the RAD52 in the MCF-7 cell may be the target gene of the let-7b, And let-7b negatively regulate the expression of the RAD52 protein.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9
【参考文献】
相关会议论文 前1条
1 贾稳;刘招舰;龚瑶琴;邵常顺;;miR-221过表达能够下调Rad51水平并促进肿瘤细胞对化疗药物的敏感性[A];第十二次全国医学遗传学学术会议论文汇编[C];2014年
相关博士学位论文 前1条
1 刘宏亮;DNA损伤修复及叶酸代谢基因单核苷酸多态与中国人群肺癌遗传易感性研究[D];复旦大学;2007年
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