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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 effect of PKD3 on the expression of prostate-specific antigen (PSA) in prostate cancer cells and its mechanism in order to clarify the important role of PKD3 in the proliferation of androgen-dependent prostate cancer cells. Methods Firstly, PKD3 was overexpressed in LNCaP cells and stimulated with dihydrotestosterone. Then the PSA cDNA was amplified by fluorescence quantitative PCR and the relative expression of PSA was compared using 2-ΔΔCt method. Secondly, in HEK293 cells, Co-transfection of PKD3 wild-type plasmid (or mutant kinase without kinase activity), AR expression plasmid, AR transcriptional reporter plasmid pMMTV-luc and internal reference reporter plasmid pRL-SV40, and stimulated with dihydrotestosterone, AR transcriptional activity was measured using Promega’s Dual Reporter Assay Kit. Finally, confocal microscopy was used to analyze changes in subcellular localization and possible co-localization of endogenous PKD3 and AR in LNCaP cells in the presence or absence of dihydrotestosterone stimulation. Results In LNCaP cells, overexpression of PKD3 significantly increased PSA mRNA expression (P <0.001) when dihydrotestosterone was stimulated. In contrast, overexpression of PKD3 significantly increased the transcriptional activity of AR (P <0.001) in HEK293 cells, while over-expression of PKD3 did not result in a decrease in AR transcriptional activity (P <0.01). In addition, endogenous PKD3 and AR in LNCaP cells both translocate to the nucleus and co-localize in the nucleus upon dihydrotestosterone stimulation. Conclusions PKD3 enhances the transcriptional activity of AR in prostate cancer cells stimulated by dihydrotestosterone and up-regulates the expression of PSA, suggesting that PKD3 may be involved in the growth and malignant proliferation of androgen-dependent prostatic cells.