钙蛋白酶2通过诱导三阴乳腺癌细胞上皮间质转化抵抗紫杉醇的机制探讨

Mechanism of Calpain-2 resistance to paclitaxel through induction of epithelial mesenchymal transition in triple-negative breast cancer cells

目的观察钙蛋白酶2(Calpain-2)在三阴乳腺癌细胞抵抗紫杉醇(PTX)中的作用及机制。方法人三阴性乳腺癌MDA-MB-231细胞转染Calpain-2质粒及相应空载体,设为过表达组和空载体组,同时以未转染细胞作为空白组。MTT法检测PTX对各组细胞存活率的影响;免疫荧光实验检测YAP的分布情况;蛋白质免疫印迹实验检测Calpain-2、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、磷酸化大肿瘤抑制基因1(pLATS1)及磷酸化yes相关蛋白(p-YAP)表达。在过表达Calpain-2的基础上加用YAP抑制剂XMU-MP-1进行干预,观察YAP对PTX抵抗的介导作用。结果与空载体组相比,PTX处理后过表达组细胞存活率增加,半数抑制浓度(IC50)升高(P<0.05)。与空载体组相比,过表达组细胞N-cadherin和Vimentin蛋白表达上调,E-cadherin蛋白表达下调(P<0.05),p-LATS1和p-YAP蛋白表达上调(P<0.01),YAP在细胞核内分布减少。与过表达组相比,XMU-MP-1+过表达组N-cadherin和Vimentin蛋白表达下调,E-cadherin蛋白表达上调(P<0.01)。PTX处理后,与过表达组相比,XMU-MP-1+过表达组细胞存活率减少,IC50降低(P<0.01)。结论Calpain-2通过YAP诱导三阴乳腺癌细胞上皮间质转化,从而对PTX产生药物抵抗。

Objective To investigate the role and mechanism of Calpain-2 in paclitaxel(PTX)resistance in triple negative breast cancer cells.Methods Human triple negative breast cancer MDA-MB-231 cells were transfected with Calpain-2 plasmid and corresponding empty vector,and cells were used as the overexpression group and the empty vector group.The untransfected cells were used as the blank group.MTT assay was performed to detect the effect of PTX on the survival rate of each group of cells.Western blot assay was performed to detect the expression of Calpain-2,E-calmodulin(E-cadherin),N-cadherin,Vimentin,phosphorylated large tumour suppressor gene 1(p-LATS1)and phosphorylated yes associated protein(p-YAP).Immunofluorescence assay was performed to detect the distribution of YAP.YAP inhibitor XMU-MP-1 was added to intervene on the basis of overexpression of Calpain-2.The mediated effect of YAP on PTX resistance was observed.Results Compared with the empty vector group,the survival rate of cells increased after PTX treatment in the overexpression group,and the IC50 value increased(P<0.05).Compared with the empty vector group,N-cadherin and Vimentin protein expression were up-regulated,E-cadherin protein expression was down-regulated in the overexpression group(P<0.05),and p-LATS1 and p-YAP protein expression were up-regulated(P<0.01).YAP distribution in nucleus was reduced in the overexpression group.Compared with the overexpression group,N-cadherin and Vimentin protein expression were down-regulated and E-cadherin protein expression was up-regulated in the XMU-MP-1+overexpression group(P<0.01).After PTX treatment,cell survival was reduced,and IC50 was lower in the XMU-MP-1+overexpression group compared with the overexpression group(P<0.01).Conclusion Calpain-2 induces epithelial mesenchymal transition in triple-negative breast cancer cells via YAP,resulting in drug resistance to PTX.