内皮素-1活化ROCK/SLUG诱导人卵巢癌细胞发生上皮向间充质转分化

Endothelin-1-induced epithelial to mesenchymal transition in human ovarian cancer cells via ROCK/SLUG pathway

目的研究ROCK/SLUG信号通路在内皮素-1(endo-thelin-1,ET-1)促进人卵巢癌细胞上皮向间充质转分化(epi-thelial to mesenchymal transition,EMT)中的作用。方法 ET-1处理人卵巢癌细胞株SK-OV-3和CaOV3,或共用ROCK的活化突变体转染细胞或加入ROCK的抑制剂Y27632,并转染含SLUG启动子的pGL3质粒与Renilla质粒。实验末用Boyden小室体外侵袭实验检测细胞侵袭能力,细胞免疫荧光染色观察细胞形态,报告基因检测试剂盒检测SLUG启动子活性,用实时定量PCR和Western bot方法检测EMT相关基因的表达。结果 ET-1诱导SK-OV-3和CaOV3发生与EMT相一致的形态和基因变化,促进其细胞侵袭力;ET-1与内皮素A受体(endothelin A receptor,ETAR)结合,促进转录因子SLUG的转录;ET-1促进ROCK及fibronectin的表达,同时转染ROCK的活化突变体,促进ET-1诱导的fibronectin表达以及细胞侵袭力的增加。相反,ROCK抑制剂Y27632抑制ET-1对fibronectin表达以及细胞侵袭力的促进作用;转染ROCK的活化突变体,上调SLUG基因转录启动子活性促进其转录,抑制E-cadherin的转录。相反,ROCK的抑制剂Y27632抑制SLUG基因启动子的活性。结论 ET-1通过活化ROCK/SLUG通路促进人卵巢癌细胞发生EMT。

Aim To study the role of ROCK/SLUG signaling pathway in endothelin-1（ET-1） induced epithelial to mesenchymal transition（EMT） in human ovarian cancer cells.Method ET-1 was used to treat SK-OV-3 and CaOV3 cells,and the cells were transfected with constitutively active mutant of ROCK,or the cells were added with ROCK inhibitor（Y27632）,or the cells were transfected with SLUG promoter-pGL3 construct and Renilla plasmid.At the end of experiment,Boyden Chamber invasion assay was used to detect the cells invasive capacity,and immunofluorescence stains were employed to observe the cells morphologic change,and reporter gene assay kit was adopted to detect SLUG promoter activity,and real time PCR and western blot were used to detect the expression of some genes related with EMT.Result ET-1 induced morphologic and genetic changes were consistent with EMT in SK-OV-3 and CaOV3 cells,and increased the invasive capacity of the cells;ET-1 combined with its receptor endothelin A receptor（ETAR） increased the transcription of SLUG;ET-1 increased the expression of ROCK and fibronectin,and when the cells were con-transfected with constitutively active mutant of ROCK,the enhancement of fibronectin and the invasive capacity of cells were increased further.On the contrary,ROCK inhibitor Y27632 inhibited the increase of fibronectin and invasive capacity of the cells induced by ET-1;when the cells were transfected with constitutively active mutant of ROCK,the SLUG promoter activity was enhanced and then the transcription of SLUG was increased,the transcription of E-cadherin was reduced.On the contrary,ROCK inhibitor Y27632 inhibited SLUG promoter activity.Conclusion ET-1 induces epithelial to mesenchymal transition in human ovarian cancer cells via ROCK/SLUG signaling pathway.