【摘要】：乳腺癌是女性最常見的惡性腫瘤之一,近年來其發(fā)病率呈不斷上升的趨勢,且發(fā)病年齡日益趨于年輕化,嚴(yán)重影響了女性的身心健康。目前研究認(rèn)為乳腺癌的發(fā)生發(fā)展主要是女性生殖因素和遺傳因素共同作用的結(jié)果,多項全基因組關(guān)聯(lián)研究(Genome Wide Association Study,GWAS)發(fā)現(xiàn)同源重組修復(fù)(Homology Recombination Repair,HRR)通路基因單核苷酸多態(tài)性(SNP)是個體DNA損傷修復(fù)能力差異的分子遺傳學(xué)基礎(chǔ),mi RNAs種子區(qū)以堿基互補(bǔ)配對方式與HRR通路靶基因m RNA 3’非翻譯區(qū)(3’UTR)特異結(jié)合,導(dǎo)致m RNA降解或抑制其翻譯從而影響HRR通路基因的表達(dá)和功能,進(jìn)而DNA損傷修復(fù)能力下降或缺失等引發(fā)的基因組不穩(wěn)定是乳腺癌發(fā)生的重要原因。目的探討HRR通路mi RNA靶基因單核苷酸多態(tài)性(MRE11A rs2155209、NBS1rs2735383、RAD51 rs963917和rs963918及RAD52 rs7963551)與乳腺癌遺傳易感性的關(guān)聯(lián)以及與女性生殖因素的交互作用,為乳腺癌的個體化預(yù)防提供理論依據(jù),且對篩選出的mi RNA let-7b進(jìn)行功能實驗驗證,為mi RNA對HRR通路靶基因的調(diào)控機(jī)制提供依據(jù)。方法(1)結(jié)合國內(nèi)外文獻(xiàn)數(shù)據(jù)庫,采用生物信息學(xué)方法及相關(guān)mi RNA靶基因預(yù)測軟件篩選HRR通路mi RNA靶序列SNPs;(2)采用病例對照研究方法,在河南漢族人群中選取醫(yī)院來源的乳腺癌病例450例,按年齡進(jìn)行頻數(shù)匹配選擇社區(qū)來源的健康對照450例,采用PCR-RFLP和AS-PCR方法對五個位點進(jìn)行基因分型檢測,使用SPSS 21.0軟件并進(jìn)行t-檢驗、卡方檢驗和非條件logistic回歸模型分析,利用Hardy-weinberg平衡在線分析軟件判斷對照組是否具有人群代表性及SHEsis在線軟件進(jìn)行RAD51基因單體型分析,應(yīng)用MDR 2.0軟件分析基因-生殖因素交互作用;(3)采用分子克隆技術(shù)構(gòu)建let-7b過表達(dá)載體,結(jié)合let-7b抑制劑分別轉(zhuǎn)染到乳腺癌細(xì)胞系MCF-7和SKBR3內(nèi),采用q RT-PCR分析let-7b和m RNA RAD52的相對表達(dá)量及Western-blot分析RAD52蛋白表達(dá)水平進(jìn)而判斷RAD52是否是let-7b的靶基因。結(jié)果(1)單位點分析結(jié)果顯示:MRE11A rs2155209 TC(OR調(diào)整=1.87,95%CI=1.23-2.86)和TC+CC(OR調(diào)整=1.86,95%CI=1.23-2.80)基因型可增加乳腺癌發(fā)病風(fēng)險,RAD52 rs7963551 AC(OR調(diào)整=0.67,95%CI=0.48-0.87)、CC(OR調(diào)整=0.36,95%CI=0.24-0.58)和AC+CC(OR調(diào)整=0.71,95%CI=0.59-0.82)基因型可降低乳腺癌發(fā)病風(fēng)險;(2)分層分析結(jié)果顯示: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)顯著增加乳腺癌發(fā)病風(fēng)險,rs2735383 GC+GG基因型在未絕經(jīng)女性中可顯著降低乳腺癌發(fā)病風(fēng)險(OR=0.47,95%CI=0.33-0.66),rs963918AG+GG基因型在生育次數(shù)≤2次的女性中乳腺癌發(fā)病風(fēng)險較高(OR=1.64,95%CI=1.10-2.45),rs7963551 AG+GG基因型在母乳喂養(yǎng)女性中的乳腺癌發(fā)病風(fēng)險較高(OR=1.49,95%CI=1.13-1.96),在PR陽性乳腺癌患者中,rs7963551 AC+CC(OR=1.82,95%CI=1.33-2.48)基因型均顯示與較高的乳腺癌發(fā)病風(fēng)險相關(guān);(3)五個位點聯(lián)合效應(yīng)分析結(jié)果顯示:隨著突變位點個數(shù)的增加,乳腺癌的發(fā)病風(fēng)險呈現(xiàn)增加的趨勢(Ptrend=0.003);(4)單體型分析結(jié)果顯示:RAD51基因Trs963917Ars963918和Trs963917Grs963918單體型可降低乳腺癌發(fā)病風(fēng)險(OR調(diào)整=0.53,95%CI=0.4-0.68),而Trs963917Ars963918和Trs963917Grs963918單體型與乳腺癌發(fā)病風(fēng)險增加相關(guān)(OR調(diào)整=1.28,95%CI=1.05-1.57和OR調(diào)整=1.31,95%CI=1.09-1.62);(5)基因-生殖因素交互作用顯示:攜帶rs2155209位點C等位基因和rs7963551位點A等位基因及具有乳腺癌家族史和母乳喂養(yǎng)經(jīng)歷的“高�！迸匀橄侔┌l(fā)病風(fēng)險是非上述組合的“低�！迸缘�3.59倍;(6)單雙酶切電泳及測序結(jié)果顯示:p Genesil-1-let-7b過表達(dá)載體構(gòu)建成功;q RT-PCR結(jié)果顯示在MCF-7細(xì)胞和SKBR3細(xì)胞中,let-7b和m RNA RAD52在let-7b過表達(dá)組和抑制劑組中的相對表達(dá)量差異均具有統(tǒng)計學(xué)意義(P0.05);(7)Western-blot結(jié)果顯示:在MCF-7細(xì)胞中,let-7b過表達(dá)組中RAD52蛋白表達(dá)水平下調(diào),let-7b抑制劑組中RAD52蛋白表達(dá)水平上調(diào);在SKBR3細(xì)胞中,RAD52蛋白在let-7b過表達(dá)組和抑制劑組中的表達(dá)量差異均無統(tǒng)計學(xué)意義(P0.05)。結(jié)論(1)MRE11A rs2155209 TC/TC+CC基因型可能增加乳腺癌發(fā)病風(fēng)險,RAD52rs7963551 C突變等位基因可能降低乳腺癌發(fā)病風(fēng)險,RAD51基因單體型CA、TA和TG與乳腺癌遺傳易感性相關(guān),rs2155209、rs7963551、母乳喂養(yǎng)和乳腺癌家族史在乳腺癌發(fā)生中具有交互作用;(2)p Genesil-1-let-7b過表達(dá)載體成功轉(zhuǎn)染到人乳腺癌細(xì)胞MCF-7和SKBR3內(nèi),并在此兩種細(xì)胞中成功上調(diào)了let-7b的表達(dá)水平,在MCF-7細(xì)胞中RAD52可能是let-7b的靶基因,且let-7b負(fù)性調(diào)節(jié)RAD52蛋白的表達(dá)。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R737.9

【參考文獻(xiàn)】

相關(guān)會議論文 前1條

1 賈穩(wěn);劉招艦;龔瑤琴;邵常順;;miR-221過表達(dá)能夠下調(diào)Rad51水平并促進(jìn)腫瘤細(xì)胞對化療藥物的敏感性[A];第十二次全國醫(yī)學(xué)遺傳學(xué)學(xué)術(shù)會議論文匯編[C];2014年

相關(guān)博士學(xué)位論文 前1條

1 劉宏亮;DNA損傷修復(fù)及葉酸代謝基因單核苷酸多態(tài)與中國人群肺癌遺傳易感性研究[D];復(fù)旦大學(xué);2007年

，

本文編號：2508705