CDH1基因1018位点突变对乳腺癌非同源末端连接修复途径的影响及作用机理研究  

Effect of CDH1 gene 1018 site mutation on non-homologous end joining repair pathway in breast cancer and its mechanism

作　　者：汤永喆 王杰[1] 何奇[1] TANG Yongzhe;WANG Jie;HE Qi(Department of Breast Diseases,Intemational Peace Maternity and Child Hospital,Shanghai Jiao Tong University,Shanghai 200030,China)

机构地区：[1]上海交通大学医学院附属国际和平妇幼保健院乳腺科,上海200030

出　　处：《中国癌症杂志》2018年第10期749-754,共6页China Oncology

基　　金：上海交通大学医工(理)交叉基金(YG2015MS43);上海交通大学医学院附属国际和平妇幼保健院院内基金

摘　　要：背景与目的:乳腺癌是女性最常见的恶性肿瘤。发现并排查乳腺癌的致病突变基因,可能达到防治乳腺癌的目的。本文旨在研究上皮钙黏蛋白编码基因(E-cadherin gene,CDH1)及其1018位点突变型在DNA损伤修复模型中的作用。方法:检测乳腺癌先证者及其家系成员血液DNA样本,发现CDH1基因的1018位错义突变c.1018A>G(p.Thr340Ala)符合遗传学定律,疑似致病突变。在此基础上,构建CDH1基因敲除的MDA-MB-231乳腺癌细胞系,将构建的CDH1-1018突变质粒与非同源末端连接(non-homologous end joining,NHEJ)报告系统共同转染进入细胞中,通过MTT法检测细胞增殖,采用流式细胞术检测NHEJ报告系统荧光率,反映CDH1基因1018位点突变对DNA损伤修复的影响,最后采用蛋白质印迹法(Western blot)对NHEJ修复途径中及乳腺癌相关的关键蛋白进行分子验证。结果:采用CRISPR/cas9系统对CDH1基因进行敲除,成功构建CDH1基因敲除细胞系;将NHEJ报告系统与CDH1基因质粒及CDH1-1018位突变质粒转染进入细胞,经i-sceⅠ酶切,测得CDH1基因1018位突变体细胞株在DNA损伤修复过程中的效率明显降低,Western blot验证了该突变对NHEJ途径重要蛋白以及乳腺癌相关蛋白表达均有抑制作用;MTT实验表明CDH1基因1018位点突变后,细胞增殖效率减缓。结论:CDH1基因1018位点突变对NHEJ修复途径有抑制作用,其机制可能与细胞粘连及组织运动能力相关,抑制相关蛋白的招募过程,使表达减弱。Background and purpose： Breast cancer is one of the most common malignant tumors in women. The discovery of a mutation gene in breast cancer may help to prevent and treat breast cancer. This article aimed to study the effect of E-cadherin gene （CDH1） and its 1018 site mutant in a DNA damage repair model. Methods： Detection of blood DNA samples from patients with breast cancer and their families revealed a missense mutation c. 1018A〉G （p.Thr340Ala） at the site 1018 of CDH1 gene which was suspected to be a pathogenic mutation. The CDH1 gene knockout MDA-MB-231 breast cancer cell lines were constructed. The constructed CDHI-IO18 site mutaion plasmid and the non-homologous end joining （NHEJ） reporting system were transfected into cells. Cell proliferation was detected by MTT assay. Fluorescence rate of NHEJ reporting system was detected by flow cytometry. Western blot assay was used for molecular verification of key proteins related to breast cancer and NHEJ repair pathway. Results： The CDH1 gene knockout MDA-MB-231 breast cancer cell lines were established using CRISPR/Casg. After i-see 1 enzyme digestion, NHEJ reporter system that transfected with CDH1-1018 site mutaion plasmid showed significantly reduced efficiency. Western blot assay confirmed that the mutation inhibited the expression of important proteins in NHEJ pathway and breast cancer-related proteins. MTT assay indicated that cell proliferation efficiency decreased after transfection of CDH1-1018 site mutation plasmid. Conclusion： CDHI gene 1018 site mutation has an inhibitory effect on the NHEJ repair pathway, and its mechanism may be related to cell adhesion and tissue movement ability, thus inhibiting the recruitment process of relevant proteins and reducing the expression.

关 键 词：遗传性乳腺癌 上皮钙黏蛋白编码基因 CRISPR/Cas9 非同源末端连接 流式细胞术 

分 类 号：R737.9[医药卫生—肿瘤]