摘要
目的观察乳腺癌细胞MCF-7发生他莫昔芬耐受后细胞形态的变化及上皮-间质转化(epithelial-mesenchymal transition,EMT)现象,并初步研究其机制。方法免疫荧光染色观察乳腺癌细胞MCF-7及其他莫昔芬耐药株MCF-7R细胞的形态;qRT-PCR、Western blot检测两种细胞EMT相关标志蛋白的表达;Transwell法检测两种细胞的迁移能力;分别抑制PI3K-Akt及MAPK/Erk信号通路,检测MCF-7R细胞的迁移能力及EMT表型的变化。结果 MCF-7细胞间的联系紧密,发生他莫昔芬耐受后,细胞间隙明显增大;MCF-7细胞发生他莫昔芬耐受后迁移能力明显增强,并发现E-钙粘素(E-cadherin)分布从细胞膜向细胞质转移,波形蛋白(vimentin)及纤维粘连蛋白(fibronectin)表达水平明显升高(P<0.05);在耐药细胞中PI3K-Akt及MAPK/Erk信号通路均处于异常活化状态,抑制PI3K-Akt信号通路能明显抑制细胞的迁移能力,同时能使波形蛋白和纤维粘连蛋白表达水平降低(P<0.05)。结论乳腺癌MCF-7细胞发生他莫昔芬耐受后其迁移能力增强可能与其发生EMT样表型有关。
Objective To investigate the changes of the cell morphology and the expression of epithelial-mesenchymal transition（ EMT） marker proteins after MCF-7 cells acquired tamoxifen resistance.Methods The cell morphology was revealed through immunofluorescent staining. The expression of EMT marker proteins was detected by quantitative real-time PCR（ qRT-PCR） and Western blotting. Transwell assay was used to detect the difference of cell migration. PI3K-Akt and MAPK / Erk signaling pathways were evaluated in MCF-7 cells and tamoxifen-resistant MCF-7R cells. The migration and the expression of EMT marker proteins were reevaluated after inhibiting the signaling pathways. Results In the MCF-7 cells,intercellular contact was close and E-cadherin was predominantly localized in the membrane. However,cell contact became loose and E-cadherin was almost translocated to the cytoplasm in the tamoxifen-resistant MCF-7R cells. The migration was enhanced and the expression of vimentin and fibronectin was increased when the MCF-7 cells acquired tamoxifen resistance（ P〈0. 05）. PI3K-Akt and MAPK / Erk signaling pathways were abnormally activated in the tamoxifen-resistant MCF-7R cells. Meanwhile,the migration and the expression of mesenchymal marker proteins were significantly suppressed when inhibiting PI3K-Akt pathway rather than MAPK / Erk pathway（ P〈0. 05）. Conclusion EMT may contribute to the increased migration of tamoxifen-resistant breast cancer cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2014年第22期2272-2276,共5页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81072149)~~
关键词
乳腺癌
他莫昔芬耐受
上皮-间质转化
细胞迁移
breast cancer
tamoxifen resistance
epithelial-mesenchymal transition
cell migration
