卵巢癌细胞放疗抗性与上皮–间质转化的关系

The relationship between radio-resistance of ovarian cancer cells and epithelial-mesenchymal transition

目的 探究卵巢癌细胞放疗抗性与上皮-间质转化(epithelial-mesenchymal transition,EMT)的关系。方法 构建A2780卵巢癌细胞放疗抵抗模型A2780R。采用平板克隆形成实验、CCK8实验检测细胞的放疗抵抗性,采用流式细胞术检测细胞凋亡,采用细胞划痕和Transwell侵袭迁移实验检测细胞迁移侵袭能力,采用Western blot和q RT-PCR实验检测细胞EMT相关蛋白和m RNA的表达。结果 与A2780细胞比,A2780R细胞的克隆体积大、数量多,细胞伸出伪足,与EMT的典型细胞形态类似。与A2780细胞比,A2780R细胞2 Gy细胞存活分数(survival fraction,SF2)、平均致死剂(mean lethal dose,D0)、准阈量(quasithreshold dose,Dq)、N增加(P <0.05),D0放射增敏比(sensitization enhancement ratio of D0,SERD0)、准阈量放射增敏比(sensitization enhancement ratio of Dq,SERDq)降低(P <0.05)。与A2780比,A2780R的凋亡率,E-钙黏蛋白(E-cadherin)蛋白与m RNA表达减少,24 h和48 h细胞迁移、24 h穿过Transwell膜和Matrigel胶细胞数量,钙黏蛋白(N-cadherin)蛋白与m RNA、波形蛋白(Vimentin)蛋白与m RNA、Snail、p-Akt、p-GSK-3β蛋白增加(P <0.05)。结论 卵巢癌放疗抵抗细胞株A2780R发生EMT,其可能机制与激活Akt/GSK-3β/Snail信号通路相关。

Objective To explore the relationship between radio-resistance of ovarian cancer cells and epithelial-mesenchymal transition(EMT).Methods A2780R,a radio-resistance model of A2780 ovarian cancer cells,was constructed.The plate clone formation assay and CCK8 assay were used to detect the radio-resistance of cells.The flow cytometry was used to detect the apoptosis of cells.The western blot and qRT-PCR experiments were used to detect the expression of EMT-related proteins and mRNAs in cells.Results Compared with A2780 cells,A2780R cell clones were larger and more numerous,and the cells protruded pseudopodia,which suggested A2780R cells were similar to the typical cell morphology of EMT.Compared with A2780 cells,the 2 Gy cell survival fraction(SF2),mean lethal dose(D0),quasi-threshold dose(Dq),N of A2780R cells were significantly increased(P <0.05),while the sensitization enhancement ratio of DO(SERDO) and quasi-threshold radiation sensitization ratio(sensitization enhancement ratio of Dq,SERDq)were significantly decreased(P<0.05).Compared with A2780,the apoptotic rate,E-cadherin protein and mRNA in A2780R of A2780R were decreased(P <0.05),while the migration distance of A2780R cells at 24 h and 48 h,the number of cells that passed through Transwell membrane and Matrigel gel at 24 h,the expression of N-cadherin protein and mRNA,Vimentin protein and mRNA,Snail,p-Akt and p-GSK-3p protein of A2780R cells were increased(P<0.05).Conclusion The EMT of ovarian cancer radiotherapy-resistant cell line A2780R may be related to the activation of Akt/GSK-3β/Snail signaling pathway.