MAPK信号通路对人胆管癌细胞Fas配体基因表达的调节及其机制的初步探讨.docVIP

MAPK信号通路对人胆管癌细胞Fas配体基因表达的调节及其机制的初步探讨 段世刚，李大江，陈 龙，刘子佩，王曙光 (400038 重庆 第三军医大学西南医院全军肝胆外科研究所) 【摘 要】 目的 观察MAPK/ERK kinase(MEK)抑制剂PD98059对人胆管癌细胞Fas配体（Fas ligand, FasL）表达的影响及其作用机制。方法 用RT-PCR和Western bolt检测PD98059处理组及未处理组人胆管癌细胞（QBC939）中c-Myc、p-c-Myc和FasL的表达；构建含启动子的荧光素酶报告质粒,细胞荧光素酶活性）启动子启动子荧光素酶活性启动子Fas ligand expression in human cholangiocarcinoma cells and its mechanism DUAN Shi-gang, LI Da-jiang, CHEN Long, LIU Zi-pei, WANG Shu-guang（Chinese PLA Hepatobiliary Surgery Institute, Southwest Hosp ital, Third Military Medical University, Chongqing 400038, China） 【Abstract】 Objective To observe the effect of PD98059, an inhibitor of MAPK/ERK kinase(MEK), on Fas ligand (FasL) expression in human cholangiocarcinoma cells (QBC-939) and to study its mechanism. Methods QBC-939 cells were pretreated with PD98059 (20 μM) or DMSO (vehicle) for 4 h and incubated with RIMP1640 for 24 h. Then, expression of FasL, c-Myc and phosphorylated c-Myc was detected by Western blotting and RT-PCR, and compared with that of parent QBC-939 cells not treated with PD98059. GAPDH mRNA served as an internal control. In addition, QBC-939 cells were transfected with luciferase reporter constructs of the FasL promoter (FasL-P1288), treated with PD98059 or DMSO for 4 h and incubated with RIMP1640 for 24 h. Dual-luciferase activity level was measured using the dual-luciferase reporter assay system. Finally, after treatment with PD98059, chromatin immunoprecipitation assay was performed. Chromatin-bound DNA selected by anti-p-c-Myc (Ser-62) was amplified with FasL-specific primers. Results PD98059, a specific MAPK-ERK cascade inhibitor, significantly attenuated the phosphorylation of c-Myc on Ser-62 and FasL up-regulation in QBC-939 cells. The luciferase reporter assay revealed that the FasL promoter activity level was significantly lower in cells treated with PD98059 than in those not treated with PD98059. Furthermore, chromatin immunoprecipitation assay showed that the phosphorylated c-Myc was b