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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FasL基因启动子的荧光素酶报告基因质粒,瞬时转染人胆管癌细胞,用PD98059处理后,检测荧光素酶相对活性,比较启动子活性的变化情况;染色质免疫共沉淀(ChIP)技术分析p-c-Myc和FasL基因启动子的结合情况。结果PD98059处理组胆管癌细胞磷酸化c-Myc(p-c-Myc)和FasL的表达均明显降低,而c-Myc未见明显变化;人胆管癌活细胞中p-c-Myc与FasL基因启动子有结合;PD98059处理组胆管癌细胞荧光素酶活性与对照组相比下降约80%。结论PD98059通过抑制MAPK/ERK kinase(MEK)的活性,降低c-Myc磷酸化水平,进而下调FasL基因启动子活性,从而抑制人胆管癌细胞FasL基因的表达。
Objective To investigate the effect of PD98059, a MAPK / ERK kinase (MEK) inhibitor, on the expression of Fas ligand (Fas ligand) in human cholangiocarcinoma cells and its mechanism. Methods The expression of c-Myc, pc-Myc and FasL in PD98059-treated and untreated human cholangiocarcinoma cells (QBC939) were detected by RT-PCR and Western blot. The luciferase reporter plasmid containing FasL gene promoter was constructed, The transient transfection of human cholangiocarcinoma cells with PD98059 treatment, the detection of luciferase relative activity, compare the promoter activity changes; ChIP assay pc-Myc and FasL gene promoter binding. Results The expression of phosphorylated c-Myc (pc-Myc) and FasL in PD98059-treated cholangiocarcinoma cells were significantly decreased, while there was no significant change in c-Myc; pc-Myc in live cholangiocarcinoma cells combined with FasL promoter ; PD98059 treatment group cholangiocarcinoma cell luciferase activity decreased by about 80% compared with the control group. Conclusion PD98059 can inhibit the expression of FasL gene in human cholangiocarcinoma cells by inhibiting the activity of MAPK / ERK kinase (MEK), decreasing the phosphorylation of c-Myc, and then down-regulating the promoter activity of FasL gene.