乳腺癌中DKKL1的表达、作用及机制探讨  

Expression and effects of DKKL1 in breast cancer and its mechanism

作　　者：王丁[1] 甘霖[2] 张国铎[1] 刘莉[3] 陈艳[3] WANG Ding;GAN Lin;ZHANG Guoduo;LIU Li;CHEN Yan(Department of General Surgery,Chongqing Traditional Hospital,Chongqing 400011,China;Department of Thyroid and Breast Surgery,Chongqing Traditional Hospital,Chongqing 400011,China;Depar tment of S urgic al Anesthesiology,Chongqing Traditional Hospital,Chongqing 400011,China)

机构地区：[1]重庆市中医院普外科,重庆400011 [2]重庆市中医院甲状腺乳腺外科,重庆400011 [3]重庆市中医院手术室,重庆400011

出　　处：《肿瘤》2020年第10期696-707,共12页Tumor

基　　金：重庆市科研机构绩效激励引导专项(编号:cstc2018jx1130063)。

摘　　要：目的:研究Dickkopf样蛋白1(dickkopf-like protein 1,DKKL1)基因在乳腺癌组织和细胞系中的表达水平,探讨DKKL1基因过表达对人乳腺癌MDA-MB-231和MCF-7细胞增殖、迁移的影响及其分子作用机制。方法:采用实时荧光定量PCR检测乳腺癌细胞系、80例人乳腺癌和癌旁组织以及20例正常乳腺组织中DKKL1 mRNA的表达水平;免疫组织化学法检测人乳腺癌组织中DKKL1蛋白的表达水平并分析与患者临床病理特征的相关性。选取表达水平最低的MDA-MB-231和MCF-7细胞,分别转染空载体pcDNA3.1(+)-Flag质粒(作为对照组)和重组pcDNA3.1(+)-Flag-DKKL1质粒(作为实验组)后,采用蛋白质印迹法验证DKKL1蛋白是否过表达;然后分别采用CCK-8法、软琼脂克隆形成实验、吖啶橙/溴化乙锭双染实验和Transwell小室迁移实验检测MDA-MB-231和MCF-7细胞增殖和迁移的变化;并采用蛋白质印迹和实时荧光定量PCR法分别检测DKKL1过表达对上皮-间质转化及下游靶分子表达和Wnt/β-连环蛋白(β-catenin)通路的影响。结果:人乳腺癌细胞系MDA-MB-231、MCF-7、BT549、MDA-MB-468、T47D和ZR-75-1中DKKL1 mRNA表达水平均明显低于正常乳腺上皮细胞MCF-10A(P值均<0.001)。与20例正常乳腺组织和80例配对癌旁组织相比,人乳腺癌组织中DKKL1 mRNA和蛋白表达均明显下调(P值均<0.01);DKKL1蛋白表达与乳腺癌的淋巴结转移、组织学分级及TNM分期具有明显相关性(P值均<0.05)。DKKL1过表达质粒转染后,乳腺癌MDA-MB-231和MCF-7细胞中DKKL1蛋白的表达水平明显上调,而细胞增殖、克隆形成、凋亡和迁移能力均被明显抑制(P值均<0.001)。DKKL1过表达可上调上皮标志物E-钙黏蛋白(E-cadherin)mRNA及蛋白表达水平,同时下调波形蛋白(Vimentin)、N-钙黏蛋白(N-cadherin)、Snail、BMI-1、KLF4和CD44 mRNA及蛋白的表达水平(P值均<0.001)。DKKL1过表达后活化的β-catenin蛋白、磷酸化的糖原合成酶激酶-3β(glycogen synthase kinase 3βObjective:To investigate the expression of dickkopf-like protein 1(DKKL1)in human breast cancer tissues and cell lines,and to analyze the effects of DKKL1 overexpression on the proliferation and migration of breast cancer MDA-MB-231 and MCF-7 cell lines,and to explore the possible mechanism.Methods:The real-time fluorescent quantitative-PCR was performed to detect DKKL1 mRNA expression levels in human breast cancer cell lines and tissues,respectively.The expression of DKKL1 protein in human breast cancer tissues was detected by immunohistochemistry,and the relationship between the expression of DKKL1 protein and the clinicopathological characteristics of breast cancer patients was analyzed.The pcDNA3.1(+)-Flag(Vetor group)and pcDNA3.1(+)-Flag-DKKL1(experimental group)plasmids were transfected into breast cancer MDA-MB-231 and MCF-7 cells,then the expression of DKKL1 was detected by Western blotting.The effects of DKKL1 overexpression on the proliferation and migration of MDA-MB-231 and MCF-7 cells were investigated by CCK-8,soft agar colony formation,acridine orange/ethidium bromide double staining and Transwell chamber assay,respectively.Western blotting and real-time fluorescent quantitative-PCR were used to detect the effects of DKKL1 overexpression on the epithelial-mesenchymal transition(EMT)progress,its downstream target molecule expressions and Wnt/β-catenin pathway.Results:The expression of DKKL1 mRNA was down-regulated in MDA-MB-231,MCF-7,BT549,MDA-MB-468,T47D and ZR-75-1 as compared with the normal breast MCF-10A cells(all P<0.001).The expression level of DKKL1 mRNA as also lower in human breast cancer than those in 20 cases of normal breast tissues and 80 cases of matched cancer adjacent tissues(all P<0.01).The expression level of DKKL1 proteins were correlated with lymph node metastasis,histological grade and TNM stage(all P<0.05).After transfection with DKKL1 overexpression plasmids,the expression of DKKL1 protein in MDA-MB-231 and MCF-7 cells was significantly up-regulated,while the cell proliferat

关 键 词：乳腺肿瘤 DKKL1基因 细胞增殖 细胞运动 WNT/Β-CATENIN信号通路 

分 类 号：R737.9[医药卫生—肿瘤]