摘要目的：初步探讨转录因子c-Jun对甲氨蝶呤耐药绒毛膜癌细胞内自噬标志蛋白--微管相关蛋白1轻链3（LC3）的调节作用。方法以人绒毛膜癌细胞株JEG-3及前期构建的甲氨蝶呤耐药绒毛膜癌细胞株JEG-3/MTXR为研究对象，采用蛋白印迹法检测甲氨蝶呤药物处理后细胞内磷酸化c-Jun氨基末端激酶（p-JNK）和磷酸化c-Jun（p-c-Jun）的表达变化，以及JNK抑制剂SP600125对p-c-Jun蛋白表达的影响。RNA干扰技术沉默c-Jun基因检测甲氨蝶呤处理后JEG-3/MTXR细胞内LC3 mRNA和蛋白的表达。染色质免疫共沉淀（ChIP）法结合实时荧光定量PCR技术测定甲氨蝶呤药物作用后JEG-3/MTXR细胞内c-Jun蛋白结合于LC3启动子上的相对富集量。结果（1）甲氨蝶呤作用后JEG-3/MTXR细胞内p-JNK和p-c-Jun蛋白的相对表达量呈时间依赖性上调，作用72 h时相对表达量分别为1.16±0.18、1.99±0.20，较0 h升高（分别为0.41±0.05、0.20±0.06），分别比较，差异均有统计学意义（P<0.05）；SP600125抑制了甲氨蝶呤作用后JEG/MTXR细胞内p-c-Jun蛋白表达的上调，甲氨蝶呤作用后JEG/MTXR细胞内p-c-Jun蛋白的相对表达量为1.11±0.20，高于SP600125联合甲氨蝶呤者（0.30±0.04），两者比较，差异有统计学意义（P<0.05）。（2）针对c-Jun基因的小分子干扰RNA （siRNA）--c-Jun-siRNA抑制了甲氨蝶呤诱导的JEG/MTXR细胞内LC3 mRNA和蛋白表达的上调，甲氨蝶呤联合c-Jun-siRNA作用后JEG-3/MTXR细胞内LC3 mRNA和蛋白的相对表达量分别为1.24±0.17、0.52±0.07，低于甲氨蝶呤联合无义siRNA者（分别为3.03±0.43、1.20±0.15），分别比较，差异有统计学意义（P<0.05）。（3）甲氨蝶呤作用后JEG/MTXR细胞内c-Jun结合于LC3启动子的相对富集量是无甲氨蝶呤作用者的（2.95±0.35）倍。结论转录因子c-Jun对LC3的调节作用可能参与绒毛膜癌发生甲氨蝶呤耐药的机制。

abstractsObjective To explore the regulation mechanism of autophagy-related protein, microtubule-associated protein 1 light chain 3 (LC3), via c-Jun in methotrexate resistant human choriocarcinoma JEG-3 cell lines. Methods Human choriocarcinoma JEG-3 cell lines, and methotrexate resistant choriocarcinoma JEG-3 (JEG-3/MTXR) cell lines were used in our present study. Phosphorylation c-Jun (p-c-Jun) was evaluated after exposure to 0.02 ng/ml methotrexate for 72 hours in both cells by western blot. c-Jun gene was knockdown by small interference RNA (siRNA) in JEG-3/MTXR cells, and LC3 was evaluated by western blot and reverse transcription-PCR. The binding of LC3 promoter with c-Jun protein was detected via chromatin immunoprecipitation assay (ChIP) with or without 0.02 ng/ml methotrexate exposure. Results The results showed that p-c-Jun was up-regulated after methotrexate treatment for 72 hours (1.99±0.20, versus 0.20±0.06 at 0 hour;P<0.05) by western blot analysis in JEG-3/MTXR cell lines. Further investigation demonstrated that c-Jun-siRNA could inhibit the up-regulation of LC3 formation and after methotrexate exposure (LC3 mRNA:1.24±0.17 versus 3.03±0.43;LC3 protein:0.52±0.07 verus 1.20± 0.15; all P<0.05). The binding of LC3 promoter by c-Jun protein was up-regulated after methotrexate treatment by the method of ChIP in methotrexate resistant JEG-3/MTXR cells [(2.95 ± 0.35) times]. Conclusion Autophagy-related gene LC3 expression regulated by c-Jun protein may be involved in the effect mechanism of the development of methotrexate resistance in choriocarcinoma JEG-3 cells.