XRCC 1基因在乳腺癌中表达的临床意义及其细胞与分子机制

Clinical significance of XRCC1 gene expression in human breast cancer and its cell and molecular mechanisms

目的:探讨乳腺癌中X线修复交叉互补蛋白1(X-ray repair cross complementing 1,XRCC1)的表达及其与临床病理特征的关系,同时分析XRCC1基因过表达对人乳腺癌MB-231细胞增殖和迁移的影响及其分子机制。方法 :采用实时荧光定量PCR法检测XRCC1 mRNA在人乳腺癌细胞系和组织中的表达水平。免疫组织化学法检测XRCC1蛋白在人乳腺癌组织中的表达情况,并统计分析乳腺癌组织中XRCC1表达与患者临床病理特征的关系。转染pcDNA3.1(+)-Flag-XRCC1重组质粒使乳腺癌MB-231细胞中XRCC1的表达上调,然后采用CCK-8法和软琼脂平板克隆形成实验检测细胞增殖活性的变化,FCM法检测细胞周期和凋亡的变化,Transwell小室法检测细胞侵袭和迁移能力的变化,蛋白质印迹法检测XRCC1过表达对细胞周期、凋亡和迁移相关蛋白表达的影响。结果 :与正常乳腺上皮细胞相比,XRCC1 mRNA在大多数乳腺癌细胞系中表达水平明显下调(P值均<0.001)。与正常乳腺上皮和配对癌旁组织相比,XRCC1 mRNA和蛋白在人乳腺癌组织中均表达下调(P值均<0.001);同时XRCC1 mRNA的表达水平与乳腺癌患者预后呈正相关性(γ2=0.052,P=0.046),而XRCC1蛋白表达与肿瘤直径、淋巴结转移、组织学分级及TNM分期具有相关性(P值均<0.05)。过表达XRCC1基因后,乳腺癌MB-231细胞的增殖、克隆形成、迁移和侵袭能力均受到明显抑制(P值均<0.01),而且细胞周期被明显阻滞于G1期(P <0.001),细胞凋亡率则明显升高(P <0.001)。XRCC1过表达能促进细胞周期相关蛋白p21、p27、Bax和剪切型caspase 3,以及迁移相关蛋白E-钙黏蛋白(E-cadherin)的表达,同时明显下调细胞周期蛋白依赖性激酶4/6(cyclin-dependent kinase 4/6,CDK4/6)、细胞周期蛋白D1(cyclin D1)、Bcl-2、N-钙黏蛋白(N-cadherin)和波形蛋白(vimentin)的表达(P值均<0.001)。结论 :XRCC1在乳腺癌细胞系和组织中表达下调,其表达水平与乳腺癌患者预后呈正相关性。恢复XRCC1基因表达能抑制细胞生长、迁移和侵袭,并诱导细胞凋亡。因此,XRCC1可能作为一个潜在的抑癌基因,参与乳腺癌的发生与发展进程。

Objective:To investigate the expression of X-ray repair cross complementing 1(XRCC1)in human breast cancer and its relationship with the clinical characteristics,and to analyze the effects of XRCC1 over-expression on the proliferation and migration of breast cancer MB-231 cells and the molecular mechanism.Methods:The expression level of XRCC1 mRNA in breast cancer cell lines and human breast cancer tissues was detected by real-time fluorescent quantitative PCR.The expression of XRCC1 protein in human breast cancer tissues was detected by immunohistochemistry.The relationship between the expression of XRCC1 protein and the clinicopathological characteristics of breast cancer patients was analyzed.The pcDNA3.1(+)-Flag-XRCC1 plasmids were transfected into breast cancer MB-231 cells for the overexpression of XRCC 1 gene.Then the proliferation activity was detected by CCK-8 and soft agar plate clone formation assay.The cell cycle and apoptosis were detected by FCM method.The cell migration and invasion were detected by Transwell chamber assay.The expressions of cell cycle-,apoptosis-and migration-related proteins were detected by Western blotting.Results:The expression level of XRCC1 mRNA was significantly decreased in most breast cancer cell lines(all P<0.001).As compared with the normal mammary epithelium and the paired adjacent breast tissues,the expression levels of XRCC1 mRNA and protein were downregulated in human breast cancer tissues(all P<0.001).The expression level of XRCC1 mRNA was positively correlated with the prognosis of breast cancer patients(γ2=0.052,P=0.046),and XRCC1 protein expression was correlated with tumor diameter,lymph node metastasis,histological grade and TNM stage(all P<0.05).After the overexpression of XRCC 1 gene,the proliferation,colony formation,invasion and migration of breast cancer MB-231 cells were significantly inhibited(all P<0.01),the cell cycle was significantly blocked in G1 phase(P<0.001),and the apoptosis rate was significantly increased(P<0.001).Furthermore,the expressions of p21,p27,Bax,cleaved caspase-3 and E-cadherin were significantly upregulated(all P<0.001),while the expressions of cyclin-dependent kinase 4/6(CDK4/6),cyclin D1,Bcl-2,N-cadherin and vimentin were down-regulated(all P<0.001)in MB-231 cells with XRCC1 overexpression.Conclusion:XRCC1 expression is down-regulated in breast cancer cell lines and tissues,and its expression level is positively correlated with the prognosis of breast cancer patients.Restoring XRCC 1 gene expression can inhibit cell growth,migration and invasion,and can induce apoptosis.So XRCC1 may be a potential tumor suppressor regulating the occurrence and development of human breast cancer.