Ezrin对人舌鳞癌顺铂耐药细胞上皮-间质转化的影响

摘要

Objective To investigate the effect of Ezrin on epithelial⁃to⁃mesenchymal transition(EMT) in human tongue squamous cell carcinoma (TSCC) with cisplatin⁃resistance. Methods A MTT⁃based method was used to analyse the half maximal inhibitory concentration (IC50) values of TSCC cell line CAL27 and its cisplatin⁃resistant cell line CAL27/CDDP. Bright⁃phase microscopic was used to observe the morphological changes. Western blot was used to de⁃tect for Vimentin, E⁃cadherin and Ezrin expression. Transwell assay was used to detect for invasion. After transfected Ezrin with siRNA, Ezrin, Vimentin and E⁃cadherin expression, as well as invasion, were furthered detected in CAL 27/CDDP. Results IC50 of CAL27/CDDP increased by 6.8 fold compared to its parent cell line. CAL27 cells with polygo⁃nal morphology stimulated by cisplatin trans⁃differentiated into epithelioid structure with clustered shape and tight con⁃nection between the cells whereas CAL27/CDDP showed a mesenchymal phenotype with an elongated morphology. CAL27/CDDP showed upregulated Vimentin, downregulated E⁃cadherin and upregulated Ezrin expression, as well as in⁃creased invasion. After knocking down Ezrin with siRNA, Ezrin and Vimentin were downregulated but E⁃cadherin was upregulated in CAL27/CDDP, as well as decreased invasion. Conclusion Knocking down of Ezrin inhibits epithelial⁃to⁃mesenchymal transition and metastasis in TSCC with cisplatin⁃resistance.%目的：探讨Ezrin蛋白下调是否能影响人舌鳞癌顺铂耐药细胞CAL27/CDDP发生上皮—间质转化（epithelial⁃to⁃mesenchymal transition ，EMT）。方法以人舌鳞癌细胞株CAL27（亲本细胞）及其顺铂耐药细胞株CAL27/CDDP（耐药细胞）为研究对象，MTT检测CAL27及CAL27/CDDP的半数抑制率（half maximal inhibitory concentration ，IC50），光学显微镜下观察细胞形态学变化；蛋白印迹检测上皮间质相关标记蛋白Vimentin、E⁃cadherin的表达变化和Ezrin的表达水平，Transwell检测细胞的迁移能力。通过小干扰RNA（Ezrin⁃Si1、Ezrin⁃Si2）转染CAL27/CDDP，检测Ezrin、Vimentin与E⁃cadherin蛋白表达及细胞迁移能力的变化。结果人舌鳞癌顺铂耐药细胞CAL27/CDDP较亲本细胞CAL27 IC50提高6.8倍，差异具有统计学意义（t=5.283，P=0.0007）；CAL27细胞呈多边形上皮样结构、成簇生长、细胞之间连接紧密，CAL27/CDDP表现为单个分散的长梭形细胞，细胞间连接消失，呈现间质细胞表型；同时，相较于亲本细胞CAL27，CAL27/CDDP间质标志蛋白Vimentin表达升高（t=4.450，P=0.011），上皮标志蛋白E⁃cadherin表达降低（t=10.980，P<0.001），Ezrin蛋白表达上调（t=6.487，P=0.003），细胞的迁移能力增强（t=3.403，P=0.010）。小干扰RNA转染人舌鳞癌顺铂耐药细胞CAL27/CDDP后Ezrin蛋白表达明显降低，Vimentin表达降低，E⁃cadherin表达升高，细胞的迁移能力显著下降。结论下调Ezrin能抑制人舌鳞癌顺铂耐药细胞发生上皮—间质转化及转移。