慢病毒介导雌激素受体β在乳腺癌细胞中的高表达抑制上皮细胞间质转化相关基因的表达

Lentivirus Mediated Estrogen Receptor β Overexpression Inhibits Epithelial-Mesenchymal Transition Associated Gene Expression in Breast Cancer Cells

目的:建立稳定高表达雌激素受体β(ERβ)蛋白表达的乳腺癌ZR75-1细胞株,检测其对上皮细胞间质转化(EMT)相关基因的影响。方法:将编码人ERβ的cDNA序列插入慢病毒表达载体pCDH-EF1-MCS-T2A-Puro,将其与慢病毒包装辅助质粒共转染293T细胞后收集病毒上清,感染乳腺癌ZR75-1细胞,经嘌呤霉素筛选后获得稳定高表达含Myc标签的人ERβ的混合细胞集落,以及作为对照组整合有对照慢病毒载体pCDH-EF1-MCS-T2A-Puro的混合细胞集落,提取细胞蛋白,用Myc抗体检测Myc-ERβ融合蛋白的表达,同时检测EMT相关基因Snail、E-Cadherin、N-Cadherin的表达水平和GSK-3β的磷酸化水平。结果:建立了稳定高表达Myc-ERβ融合蛋白的乳腺癌细胞株,和对照组相比,Myc-ERβ高表达抑制EMT相关蛋白N-cadherin和Snail的表达,增强E-cadherin分子的表达,抑制GSK-3β磷酸化水平。结论:建立了Myc-ERβ高表达的乳腺癌细胞株,发现ERβ高表达抑制EMT相关分子的表达,为深入研究分子在乳腺癌中调控的机制奠定了基础。

Objective： To construct a breast cancer ZR75-1 cell line stably overexpressing estrogen receptor β（ERβ）and detect its effects on the expression of epithelial-mesenchymal transition（EMT）associated genes. Methods： Cloning ERβ cDNA into the lentivirus vector pCDH-EF1-MCS-T2A-Puro,which was then packaged with accessary plasmids into lentivirus in 293T cells. After infected with lentivirus and selected by puromycin, mixed ZR75-1 colonies stably expressing Myc-ERβ were obtained and the fusion Myc-ERβ expression was detected by Western blot. Meanwhile, the expression of EMT associated protein such as Snail, E-Cadherin, N-Cadherin and GSK-3β in ERβ overexpression ZR75-1 cells was compared with that in vector control cells. Results： N-cadherin and Snail expression was decreased and E-cadherin increased in ERβ overexpressing cells, Moreover, ERβ overexpression may decrease the GSK-3β phophorylation level. Conclusion： Breast cancer cell line overexpression ERβ was successfully constructed and ERβ overexpression may regulate the expression of several protein involved in EMT, which has established a good foundation for further study of the mechanism of the regulation of EMT by ERβ in breast cancer cells.