本文選題：乳腺癌 + 易感性��； 參考：《鄭州大學》2017年碩士論文

【摘要】：乳腺癌是女性最常見的癌癥。在中國乳腺癌還是引起中國女性癌癥死亡的首要病因。流行病學研究表明乳腺癌的發(fā)生發(fā)展通常是由遺傳、環(huán)境、生活方式和生殖危險因素的相互作用引起的。全基因組關(guān)聯(lián)研究(Genome Wide Association Study,GWAS)顯示在HRR通路上的DNA修復基因如RAD51、RAD52、XRCC2、BRCA1和BRCA2會導致乳腺癌的發(fā)生。在這些基因3’非編碼區(qū)(3’UTR)的單核苷酸多態(tài)性可以通過擾亂現(xiàn)有的或創(chuàng)造miRNAs的結(jié)合位點來調(diào)控基因的表達水平,進而導致疾病的發(fā)生。還有研究表明Linc-ROR在癌癥包括乳腺癌的發(fā)展中起著重要的作用。然而可以調(diào)節(jié)這些基因表達水平的潛在功能性單核苷酸多態(tài)性(SNP)還未被大量研究。目的本研究的目的是評估十一個SNPs和乳腺癌的遺傳易感性之間的相關(guān)性以及十一個SNPs與生殖因素之間的交互作用。此外,對于篩選出與乳腺癌有關(guān)聯(lián)性的SNP對應(yīng)的miRNA和Linc-ROR的標簽SNP進行進一步的功能驗證,從而探索miRNA調(diào)控乳腺癌發(fā)生的機制。方法(1)結(jié)合NCBI dbSNP數(shù)據(jù)庫、查閱國內(nèi)外相關(guān)文獻、預(yù)測miRNA結(jié)合位點的相關(guān)軟件以及篩選標簽SNP的heploview軟件,利用生物信息學方法篩選了HRR通路選定基因(BRCA1、BRCA2、RAD51、RAD52和XRCC2)的miRNA靶序列SNPs(rs8176318、rs12516、rs15869、rs7180135、rs45549040、rs1051669、rs3218550)以及Linc-ROR的標簽SNPs(rs1942348、rs4801078、rs6420545和rs9636089);(2)在本次病例對照研究中,根據(jù)研究功效(0.9)和最小等位基因頻率(0.25),利用PASS軟件計算樣本量為n=459。本研究納入了來自河南省的498例乳腺癌患者和按年齡(±2歲)與之進行頻數(shù)匹配的健康對照者498例。采用限制片段多態(tài)性(PCR-RFLP)和等位基因特異性(AS-PCR)的方法對研究對象的DNA樣本進行基因分型。分型結(jié)果首先進行Hardy-weinberg平衡的檢驗以保證對照人群的代表性。采用非條件logistic回歸模型分析不同基因型與乳腺癌發(fā)病風險的關(guān)聯(lián),并進一步使用多因子降維的方法分析基因與環(huán)境的交互作用。此外SHEsis在線軟件用作基因BRCA1和Linc-ROR的單體型分析。(3)miRNA-627的過表達載體與miRNA-627抑制劑分別轉(zhuǎn)染到乳腺癌細胞系MDA-MB-231與MCF-7中,利用qRT-PCR的方法來檢測不同組內(nèi)細胞miRNA-627與mRNA BRCA2的相對表達量;采用qRT-PCR的方法檢測不同基因型樣本的血漿Linc-ROR的表達水平,從而研究Linc-ROR的單核苷酸多態(tài)性對自身表達水平的影響。結(jié)果(1)BRCA1 rs8176318 GT(OR:1.332;95%CI:1.007-1.761)與GT+TT(OR:1.336;95%CI:1.018-1.753)基因型可以增加乳腺癌的發(fā)病風險;此外,BRCA1基因Crs8176318Trs12516的單體型在對照組中的頻率明顯高于病例組,而BRCA1基因Trs8176318Trs12516的單體型在對照組中的頻率顯著低于病例組。(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發(fā)病風險,分層分析顯示rs15869 AC+CC基因型在年齡大于50歲(OR:1.486,95%CI:1.047-2.109)、月經(jīng)初潮年齡≤13歲(OR:1.53,95%CI:1.07-2.188)、絕經(jīng)年齡50歲(OR:2.185,95%CI:1.094-4.366)、絕經(jīng)前(OR:1.344,95%CI:1.022-1.767)、妊娠≤2次(OR:1.639,95%CI:1.189-2.261)、流產(chǎn)≤2次(OR:1.392,95%CI:1.1-1.762)、母乳喂養(yǎng)(OR:3.576,95%CI:1.35-9.475)和無前一級親屬腫瘤家族史(OR:1.347,95%CI:1.08-1.679)的人群中增加乳腺癌的發(fā)病風險。(3)RAD51/RAD52/XRCC2 RAD51基因rs45549040 AC+CC基因型在妊娠數(shù)2次的女性人群中可以降低乳腺癌的發(fā)病風險(OR:0.631,95%CI:0.406-0.98);RAD52基因rs1051669 GA+AA基因型可以增加絕經(jīng)前女性的乳腺癌風險(OR:1.519,95%CI:1.076-2.146);XRCC2 rs3218550 GA+AA基因型在年齡小于50歲(OR:0.627,95%CI:0444-0.885)和月經(jīng)初潮年齡≤13歲(OR:0.538,95%CI:0.330-0.874)的人群中降低了乳腺癌的風險。(4)Linc-ROR rs4801078 TT(OR:1.791;95%CI:1.195-2.683)可以增加乳腺癌的發(fā)病風險;Linc-ROR單體型Trs6420545Crs4801078Trs1942348Trs9636089的頻率在對照組相對較高。(5)基因-生殖因素交互作用分析結(jié)果:攜帶rs15869位點C等位基因、初潮年齡≤13歲和懷孕次數(shù)≥2次的人群乳腺癌的發(fā)病風險是不具有這些危險因素人群的2.39倍(OR:2.39;95%CI:1.04-5.47);rs4801078、懷孕次數(shù)以及絕經(jīng)狀態(tài)三者之間的交互作用可以提高乳腺的發(fā)病風險(OR:2.78;95%CI:2.74-3.61)。(6)實時定量PCR結(jié)果:成功轉(zhuǎn)染了pGenesil-1-miRNA-627過表達質(zhì)粒的乳腺癌細胞系中BRCA2的表達水平較轉(zhuǎn)染了空質(zhì)粒的對照組顯著降低(P0.05),而成功轉(zhuǎn)染miRNA-627抑制物的兩個乳腺癌細胞系BRCA2的相對表達水平較其對照組顯著增高(P0.05);Linc-ROR的表達水平在rs4801078 CT+TT基因型組的人群中(1.94±0.55)顯著高于CC基因型組的人群(1.21±0.49)。結(jié)論(1)BRCA1 rs8176318 GT/GT+TT與BRCA2 rs15869 AC基因型可以增加乳腺癌的發(fā)病風險;(2)Linc-ROR rs4801078 TT基因型可以增加乳腺癌的發(fā)病風險;(3)攜帶rs15869位點C等位基因、初潮年齡≤13歲和懷孕次數(shù)≥2次的人群乳腺癌的發(fā)病風險是不具有這些危險因素人群的2.39倍,rs4801078、懷孕次數(shù)以及絕經(jīng)狀態(tài)三者之間的交互作用可以提高乳腺的發(fā)病風險;(4)成功轉(zhuǎn)染的pGenesil-1-miRNA-627過表達質(zhì)粒在乳腺癌細胞系MCF-7與MDA-MB-231中成功上調(diào)了BRCA2的表達水平;rs4801078 CT+TT基因型與健康對照人群中血漿Linc-ROR的表達水平顯著相關(guān)。
[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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.9

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5 葛廣哲;樹，

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