PKD1对HeLa细胞中AP1的转录激活作用  

PKD1 contributes to activator protein-1 activation in human HeLa cells

作　　者：邹志鹏[1] 许万福[1] 冯丽[1] 柯志勇[1] 邓凡[1] 

机构地区：[1]南方医科大学细胞生物学教研室,广州510515

出　　处：《重庆医学》2010年第15期1945-1947,共3页Chongqing medicine

基　　金：国家自然科学基金资助项目(30973014);教育部留学回国人员科研启动基金资助项目(K1010353);南方医科大学院长基金资助项目(JC0701)

摘　　要：目的研究蛋白激酶D1(PKD1)对人HeLa细胞中AP1转录激活作用,为探讨PKD1作用的AP1靶基因的功能作用奠定基础。方法首先将对照质粒pcDNA3.1、PKD1野生型质粒(pcDNA-PKD1)或无激酶活性突变型质粒pcDNA-PKD1-DN与AP1转录活性报告质粒pAP1-luc及内参照报告质粒pRL-SV40共转染人HeLa细胞,同时将对照siRNA(si-CTL)或PKD1 siRNA(siRNA-PKD1)与AP1转录活性报告质粒pAP1-luc及内参照报告质粒pRL-SV40也共转染人HeLa细胞。转染48h后,分别收集细胞裂解液进行Western blot检测外源性PKD1过表达或内源性PKD1敲低状况,并以Promega双报告基因分析试剂盒测定AP1荧光素酶活性,计算AP1相对转录活性。结果 Western blot证实HeLa细胞中外源性pcDNA-PKD1、pcDNA-PKD1-DN过表达,而且siRNA-PKD1明显敲低HeLa细胞中内源性PKD1表达;双色荧光素酶报告基因检测表明,与对照质粒pcDNA3.1比较,pcDNA-PKD1过表达明显增强AP1转录活性(P<0.05),相反,与pcDNA-PKD1比较,pcDNA-PKD1-DN过表达则明显降低AP1转录活性(P<0.05)。与之相符,与si-CTL比较,siRNA-PKD1对内源性PKD1敲低,也明显抑制AP1转录活性(P<0.01)。结论 PKD1表达和激酶活性增强HeLa细胞中AP1转录激活,提示PKD1可能通过AP1调控相关靶基因表达。Objective To explore functional role of PKD1 on the AP1 transcriptional activation and its targets gene in human HeLa cells. Methods pcDNA3.1,wild type of pcDNA-PKD1, kinase dead mutant of pcDNA-PKD1-DN,non specific siRNA （siCTL） or siRNA of PKDlwas eotransfected with activator protein I（AP1）luciferase reporter APl-luc and Renilla luciferase reporter pRL-SV40 into human HeLa cells. After 48 h transfection,cell lystates were harvested to measure the exogenous overexpression of wild type PKD1 and dominate negative PKD1 or endogenous PKD1 knockdown by siRNA using Western blot assay. Furthermore, AP1 transcriptional activity were determined by dual-luciferase reporter assay. Results Over-expression of PKD1 ,dominant negative mutant PKDI（PKD1-DN） or knockdown of PKD1 by siRNA in HeLa cells was confirmed by Western blot. Compared with pcDNA3.1 ,AP1 transcriptional activation triggered by PKD1 was significantly increased in HeLa cells transfeeted with peDNA- PKD1 （P〈0.05）, whereas dramatically reduced by peDNA-PKD1-DN transfection compared with wild type of peDNA-PKD1 transfection（P〈0.05）. In addition, AP1 transcriptional activation was also significantly inhibited by siRNA knockdown of endogenous PKD1 compared with control siRNA transfection（P〈0. 001）. Conclusion Enhanced AP1 transcriptional activation induced by PKD1 expression and kinase activity in HeLa cells suggested that PKD1 may regulate target genes expression through AP1 transcriptional activation.

关 键 词：蛋白激酶D1 AP1 SIRNA 转录激活 

分 类 号：R363[医药卫生—病理学]