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  

MAPK signal pathway regulates Fas ligand expression in human cholangiocarcinoma cells and its mechanism

作　　者：段世刚[1] 李大江[1] 陈龙[1] 刘子佩[1] 王曙光[1] 

机构地区：[1]第三军医大学西南医院全军肝胆外科研究所,重庆400038

出　　处：《第三军医大学学报》2010年第3期224-228,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(30772109)~~

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 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 μmol/L） or DMSO （vehicle） for4 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 lueiferase 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-speeific primers. Results PD98059, a specific MAPK-ERK cascade inhibitor, significantly attenuated the phosphorylation of e-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, ehromatin immunoprecipitation assay showed that the phosphorylated c-Myc was bond to the human FasL promoter in vivo. Conclusion PD98059 inhibits FasL expression in human cholangiocareinoma cells by inhibiting the MAPK-ERK kinase activity, decreasing the c-Myc phosphorylation level, and activating the FasL promoter.

关 键 词：转录因子 FAS配体 启动子活性 报告基因 PD98059 

分 类 号：R735.8[医药卫生—肿瘤] R394.2[医药卫生—临床医学]