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1、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/ERKkinase(MEK)抑制剂PD98059对人胆管癌细胞Fas配体（Fasligand,FasL）表达的影响及其作用机制。方法用RT-PCR和Westernbolt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
2、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/ERKkinase(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。【关键词】转录因子；Fas配体；启动子活性；报告基因；PD98059【中图法
3、分类号】【文献标志码】AMAPKsignalpathwayregulatesFasligandexpressioninhumancholangiocarcinomacellsanditsmechanismDUANShi-gang,LIDa-jiang,CHENLong,LIUZi-pei,WANGShu-guang（ChinesePLAHepatobiliarySurgeryInstitute,SouthwestHospital,ThirdMilitaryMedicalUniversity,Chongqing400038,China）【Abstract】ObjectiveToobserv
4、etheeffectofPD98059,aninhibitorofMAPK/ERKkinase(MEK),onFasligand(FasL)expressioninhumancholangiocarcinomacells(QBC-939)andtostudyitsmechanism.MethodsQBC-939cellswerepretreatedwithPD98059(20µM)orDMSO(vehicle)for4handincubatedwithRIMP1640for24h.Then,expressionofFasL,c-Mycandphosphorylatedc-Mycwasde
5、tectedbyWesternblottingandRT-PCR,andcomparedwiththatofparentQBC-939cellsnottreatedwithPD98059.GAPDHmRNAservedasaninternalcontrol.Inaddition,QBC-939cellsweretransfectedwithluciferasereporterconstructsoftheFasLpromoter(FasL-P1288),treatedwithPD98059orDMSOfor4handincubatedwithRIMP1640for24h.Dual-luc
6、iferaseactivitylevelwasmeasuredusingthedual-luciferasereporterassaysystem.Finally,aftertreatmentwithPD98059,chromatinimmunoprecipitationassaywasperformed.Chromatin-boundDNAselectedbyanti-p-c-Myc(Ser-62)wasamplifiedwithFasL-specificprimers.ResultsPD98059,aspecificMAPK-ERKcascadeinhibitor,significa
7、ntlyattenuatedthephosphorylationofc-MyconSer-62andFasLup-regulationinQBC-939cells.TheluciferasereporterassayrevealedthattheFasLpromoteractivitylevelwassignificantlylowerincellstreatedwithPD98059thaninthosenottreatedwit
