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ERβ分子在乳腺癌中调控EMT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.
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