活性氧介导P62蛋白激活参与甲氨蝶呤耐药绒癌发病机制研究

目的 探讨甲氨蝶呤（MTX）耐药绒癌的发生机制。方法 2012年5月至2013年1月于北京协和医院以人绒毛膜上皮癌JEG-3细胞及本课题组前期构建的MTX耐药绒癌JEG-3/MTXR细胞为研究对象，采用免疫印迹、RT-PCR法检测MTX处理后细胞内P62的变化。流式细胞仪检测MTX诱导后JEG-3/MTXR细胞内活性氧(ROS)变化，采用ROS清除剂MnTmPyP检测MTX作用后JEG-3/MTXR细胞内P62蛋白水平的变化。RNA干扰法沉默P62基因免疫印迹法和流式细胞仪检测MTX诱导后JEG-3/MTXR耐药细胞内PARP蛋白的表达和凋亡率的变化。结果 MTX可诱导耐药绒癌 P62蛋白和mRNA表达水平呈时间依赖性上调。MTX可诱导耐药绒癌ROS表达水平升高，MnTmPyP降低P62蛋白的表达。P62-siRNA可促进JEG-3/MTXR细胞内MTX诱导的凋亡反应蛋白PARP的切割和激活，流式细胞仪检测RNA干扰联合药物组细胞凋亡率（14.4±1.02）%，高于单纯药物应用组（9.1±1.34）%，差异有统计学意义（P＜0.05）。结论 ROS介导P62蛋白激活参与MTX耐药绒癌发生机制，为耐药绒癌临床靶向治疗提供新思路。

The effect mechanism of methotrexate resistant human choriocarcinoma JEG-3 cells via ROS-mediated JNK/P62 activation.

Abstract： Objective To explore the effect mechanism of methotrexate resistance in human choriocarcinoma JEG-3 cell lines. Methods Human choriocarcinoma JEG-3 cell lines，and methotrexate resistant choriocarcinoma JEG-3 (JEG/MTXR) cell lines were used in our present study.P62 protein and mRNA were evaluated after exposure to methotrexate (0.02 μg/L) for 72 h in both cells by western blotting or RT-PCR. Cell Oxygen Species (ROS) level was evaluated using FACS by DCFH-DA method.P62 proteins were analyzed by western blotting after exposure to ROS inhibition MnTmPyP in methotrexate treated JEG-3/MTXR cells.P62 was knockdown by P62-siRNA in MTXR/JEG-3 cell lines，and cell apoptosis was evaluated by western blotting and flow cytometry analysis.Results We found that P62 protein level was upregulated by either western blot analysis or RT-PCR in JEG-3/MTXR cell lines. Further investigation demonstrated that P62 activation was mediated by ROS，while ROS inhibitor MnTmPyP could decrease the level of P62.Silencing of P62 gene expression by siRNA facilitated the cleavage of apoptosis related protein PARP，and the upregulation of apoptotic rate （14.4±1.02 for JEG-3 vs. 9.1±1.34 for JEG/MTXR）by FACS in methotrexate-resistant choriocarcinoma JEG-3 cells.Conclusion ROS-mediated P62 activation is involved in the effect mechanism of the development of methotrexate resistance in choriocarcinoma JEG-3 cells.