Ca^(2+)/calpain信号调控纤连蛋白诱导的MCF-10A乳腺上皮细胞-间质转化的作用

Fibronectin induced epithelial-mesenchymal transition of MCF-10A mammary epithelial cells regulated by Ca^(2+)/calpain signaling

目的:观察Ca^(2+)/calpain信号通路在纤连蛋白(FN)诱导MCF-10A乳腺上皮细胞-间质转化(EMT)中的作用。方法:以正常培养MCF-10A乳腺上皮细胞为实验对象。采用比色法检测细胞内Ca^(2+)浓度;采用荧光分光光度法检测钙蛋白酶-2(calpain-2)活性;Western blot法检测calpain-2、波形蛋白(vimentin)、E-钙黏着蛋白(E-cadherin)的表达;采用划痕修复实验检测细胞迁移能力;Transwell小室实验检测细胞侵袭能力。结果:FN诱导MCF-10A细胞中Ca^(2+)浓度、calpain-2活性表达明显升高,上调vimentin、calpain-2蛋白表达,下调E-cadherin蛋白水平;FN诱导MCF-10A细胞迁移和侵袭能力显著增强。钙离子螯合剂(BAPTA-AM)和氨氯地平(amlodipine)均能显著抑制FN诱导的细胞Ca^(2+)浓度、calpain-2活性及表达升高;抑制vimentin蛋白表达上调及E-cadherin蛋白表达下调;抑制FN诱导MCF-10A细胞迁移和侵袭能力增强。结论:BAPTA-AM和Amlodipine均能够抑制FN诱导的上皮间质转化。FN能够诱导MCF-10A乳腺上皮细胞发生EMT,可能与Ca^(2+)/calpain信号通路激活有关。

OBJECTIVE To investigate the regulatory effects of Ca^（2＋）/calpain signaling pathway on epithelial-mesenchymal transition of MCF-10 A mammary epithelial cells induced by fibronectin（FN）.METHODS The MCF-10 A mammary epithelial cells were cultured in the present experiments.The cell Ca^（2＋）concentration was detected by colorimetric assay.The activity of calpain-2 was measured by fluorescence spectrophotometry.Vimentin,E-cadherin and calpain-2 protein were determined by Western blot.Cell migration was measured by wound healing assay,and invasion was determined by transwell chamber assay.RESULTS BAPTA-AM and amlodipine significantly inhibited the Ca^（2＋）and calpain-2 expression in cells induced by FN,and FN could up-regulate vimentin expression and down-regulate E-cadherin expression.The FN-induced MCF-10 Acell migration and invasion were inhibited by BAPTA-AM and amlodipine.CONCLUSION The present results show that both BAPTA-AM and amlodipine can inhibit FN-induced epithelia transformation and the mechanism may involve in Ca^（2＋）/calpain signaling pathway activation.