作　　者： ; ; ; ; ; ;

机构地区： 南方医科大学

出　　处： 《南方医科大学学报》 2010年第8期1779-1782,共4页

摘　　要： 目的研究蛋白激酶D3(PKD3)对前列腺癌细胞中前列腺特异性抗原(PSA)表达水平的影响及其机制,以期阐明PKD3在雄激素依赖的前列腺癌细胞增殖中的重要作用。方法首先,在LNCaP细胞中过表达PKD3并以双氢睾酮刺激,而后采用荧光定量PCR扩增PSA的cDNA并使用2-△△Ct方法比较其相对表达量的变化;其次,在HEK293细胞中,共转染PKD3的野生型质粒(或无激酶活性的突变型质粒)、AR的表达质粒、AR转录活性的报告质粒pMMTV-luc及内参照报告质粒pRL-SV40,并以双氢睾酮刺激后,以Promega的双报告基因分析试剂盒测定AR的转录活性。最后,利用共聚焦显微技术分析LNCaP细胞中内源性的PKD3和AR在有无双氢睾酮刺激时亚细胞定位的变化和可能的共定位。结果在LNCaP细胞中,过表达PKD3可明显升高双氢睾酮刺激时PSA的mRNA表达水平(P<0.001)。与之相符,在HEK293细胞中,过表达PKD3明显提高双氢睾酮刺激时AR的转录活性(P<0.001),而过表达无激酶活性的PKD3可部分降低AR的转录活性(P<0.01)。此外,LNCaP细胞中内源性的PKD3和AR在双氢睾酮刺激时均向核内转位并共定位于核内。结论 PKD3增强双氢睾酮刺激的前列腺癌细胞中AR的转录活性及上调PSA的表达,提示PKD3可能参与雄激素依赖的前列腺细胞的生长和恶性增殖。 Objective To investigate the role of PKD3 in prostate-specific antigen （PSA） expression regulation in androgen-dependent prostate cancer cells and explore the mechanism. Methods LNCaP cells containing low level of PKD3 were transfected with pEGFP-C2 or pEGFP-PKD3 plasmid followed by dihydrotestosterone （DHT） treatment, and PSA mRNA level was analyzed by RT-QPCR using 2-△△Ct method. Wild-type or kinase-dead PKD3 plasmids, human androgen receptor plasmid pSVAR0, pMMTV-luc of AR luciferase reporter and renilla luciferase reporter pRL-SV40 were cotransfected into HEK293 cells, and after treatment with DHT for 24 h, the cells were harvested and AR transcriptional activity were determined by dual-luciferase reporter assay. The subcellular localization of endogenous PKD3 and AR and their colocalization induced by DHT were observed by confocal microscopy. Results PSA mRNA level triggered by DHT was significantly increased by overexpression of pEGFP-PKD3 in LNCaP cells compared with that in pEGFP-C2 control cells （P〈0.001）. AR transcription in response to DHT treatment was also significantly up-regulated by wild type PKD3 expression （P〈0.001）, but partially down-regulated by kinase-dead PKD3 mutant （P〈0.01）. Endogenous PKD3 and AR in LNCaP cells not only translocated from the cytoplasm to the nucleus, but also colocalized with each other after DHT stimulation. Conclusion Elevated AR transcriptional activity and enhanced expression of PSA induced by PKD3 in response to DHT treatment suggest that PKD3 contributes to the proliferation and malignant growth of androgen-dependent prostate cancer cells.

关 键 词： 蛋白激酶 雄激素受体 前列腺癌 前列腺特异性抗原

领　　域： [生物学]