摘要
目的:构建锌指蛋白Slug基因的慢病毒真核表达载体,研究Slug对ZR75-1乳腺癌细胞上皮间质转化及迁移的影响。方法:以乳腺文库为模板,PCR扩增Slug全长编码序列,将其插入pCDH表达载体,构建pCDH-Slug真核表达载体,转染293T细胞,Western印迹检测Slug的表达;将重组质粒包装慢病毒感染ZR75-1细胞,用嘌呤霉素筛选得到稳定表达Slug的ZR75-1乳腺癌细胞,Western印迹和免疫荧光检测Slug对E-钙黏蛋白表达的影响,采用划痕实验检测Slug在ZR75-1乳腺癌细胞中对细胞迁移的影响。结果:双酶切实验证明Slug慢病毒表达载体构建成功;West ern印迹表明Slug在293T细胞内表达成功;稳定表达Slug的ZR75-1乳腺癌细胞株构建成功;镜下观察发现,稳定表达Slug的ZR75-1乳腺癌细胞形态变为梭形;Slug在ZR75-1乳腺癌细胞中可以抑制E-钙黏蛋白的表达;划痕实验发现Slug在ZR75-1乳腺癌细胞中可以促进细胞迁移。结论:构建了Slug慢病毒真核表达载体,Slug可以促进上皮间质转化,并通过调控E-钙黏蛋白表达促进细胞迁移。
Objective:To construct an eukaryotic expression vector of Slug and to detect its effect on epithelialmesenchymal transition(EMT)and the migration of ZR75-1 cells.Methods:The coding sequences of Slug were amplified from the breast library by PCR and cloned into the pCDH.The recombinant plasmid and empty vector were transfected into 293T cells,and the expression of Slug was detected by Western blot.Lentivirus was pro duced with the recombinant plasmid and to infect ZR75-1 cells,and cells were selected for 2 weeks with puromy cin before the mixed colonies stably expressing Slug were obtained.The fluorescent signals were detected by fluo rescence microscope.The effects of Slug on E-cadherin were detected by Western blot and fluorescence micro scope.Wound healing assays were performed to examine the effects of Slug on the migration of ZR75-1 cells.Results:The eukaryotic expression vector of Slug was identified by double digestion.Western blotting showed that the recombinant plasmid could express Slug protein in 293T cells.We found that the stable expression of slug in ZR75-1 breast cancer cells became fusiform by microscope.Slug could markedly inhibit the expression of E-cad herin and enhance the migration of ZR75-1 cells.Conclusion:The eukaryotic expression vector of Slug is con structed and expressed in human ZR75-1 cells.Slug can promote EMT.This study lays foundation for further re search on Slug mechanism in breast cancer cell.
作者
李华月
刘婕
韩洋
丁丽华
叶棋浓
LI Hua-Yue;LIU Jie;HAN Yang;DING Li-Hua;YE Qi-Nong(Medical School,Guizhou University,Guizhou 550025;Beijing Institute of Biotechnology,Beijing 100850;Hospital of the 81st Group Army of PLA,Zhangjiakou 075000,China)
出处
《生物技术通讯》
CAS
2020年第1期24-28,共5页
Letters in Biotechnology
基金
国家自然科学基金(31470773)。
