S期激酶相关蛋白Skp2调控雄激素受体表达及活性在去势抵抗型前列腺癌进展中的作用

Regulatory effect of Skp2 on the expression and transactivation of the androgen receptor in the progression of castration-resistant prostate cancer

目的:检测不同前列腺癌(PCa)细胞系及组织中S期激酶相关蛋白(Skp2)的表达水平,探讨其表达改变对雄激素受体(AR)信号通路及去势抵抗型前列腺癌(CRPC)发生可能的影响。方法:通过Western印迹法检测不同PCa细胞系中Skp2、AR表达;利用RNA干扰技术,转染shRNA敲低CRPC细胞系C4-2或22RV1中Skp2表达,通过Western印迹法检测雄激素处理后细胞中AR、P27表达,通过双荧光素酶报告基因法检测雄激素处理后细胞中雄激素反应元件(ARE)报告基因ARR3-Luc活性;利用免疫组化染色法检测未行内分泌治疗的患者PCa和CRPC组织标本中Skp2、AR表达,分析两者表达差异及相关性。结果:CRPC细胞系C4-2及22RV1较雄激素依赖性细胞系LNCa P中Skp2表达水平显著升高;C4-2细胞中雄激素处理可诱导Skp2表达,而shRNA敲除Skp2不仅可显著上调其下游经典靶分子P27蛋白的表达,还显著降低AR蛋白表达水平。相一致地,C4-2及22RV1细胞系中雄激素处理可增强ARR3-Luc活性,而敲除Skp2可显著抑制雄激素处理前或后的ARR3-Luc活性(P<0.05)。此外,CRPC组织较未行内分泌治疗患者PCa组织中Skp2、AR染色显著增强(P<0.05),两者表达具有正相关性(r=0.658 1,P<0.05)。结论:CRPC中Skp2可增强AR蛋白表达及转录活性,有望成为重要的分子治疗靶点。

Objective： To determine the expression of Skp2 in different prostate cancer （PCa） cell lines and tissues, and ex- plore its influence on the androgen receptor （AR） signaling pathway and development of castration-resistant prostate cancer （CRPC）. Methods ： The expression levels of Skp2 and AR in different PCa cell lines were detected by Western blot. After knockdown of Skp2 in the C4-2 and 22RV1 cells transfected with shRNA, the expressions of AR and P27 were determined and the activity of ARR3-Lue measured by dual-luciferase reporter gene assay following treatment with dihydrotestosterone （DHT）. The expressions of AR and Skp2 in human naive PCa or CRPC specimens were detected by immunohistochemical staining followed by analysis of their differences and correlation. Results ： The Skp2 protein expression level was significantly higher in the C4-2 or 22RV1 cells than in the LNCaP cells.DHT treatment increased the expression of Skp2 in the C4-2 cells, but knock-down of Skp2 significantly up-regulated the expression of the well-known downstream protein P27 and down-regulated that of AR. Consistently, DHT treatment increased the activity of ARR3- Luc, while knockdown of Skp2 remarkably decreased it in the C4-2 and 22RV1 cells （ P 〈 0.05 ）. In addition, significantly higher ex- pressions of Skp2 and AR were observed in the CRPC than in the naive specimens （ P 〈 0.05） , with a positive correlation between the two proteins （ r = 0. 658 1, P 〈 0. 05 ）. Conclusion ： Skp2 can enhance the expression and transcription activity of the AR protein in CRPC cells or tissues and is promising to be a critical molecular therapeutic target.