六价铬通过上调SUMO特异性蛋白酶家族分子表达增强雄激素受体的转录活性

Chromium Ⅵ enhances transcriptional activity of androgen receptor by upregulating SUMO-specific protease family

目的研究六价铬对雄激素受体(AR)转录活性的影响及其与小泛素类似修饰物特异性蛋白酶(SENP)家族分子的相关性。方法(1)293T细胞经K_(2)CrO_(4)0.25,0.5,1.0,2.0,4.0,8.0和16.0μmol·L^(-1)处理24 h,LNCa P细胞经K_(2)CrO_(4)0.25,0.5,1.0,2.0,4.0,10.0和20.0μmol·L^(-1)处理24 h,用MTT法检测细胞存活率并计算半数抑制浓度(IC_(50))值;(2)将带有荧光素酶报告基因的表达雄激素反应元件的质粒和表达AR的质粒共转染293T细胞,将带有荧光素酶报告基因的表达雄激素反应元件的质粒转染LNCa P细胞,分别用K_(2)CrO_(4)0.25,0.5,1.0,2.0,4.0,8.0和16.0μmol·L^(-1)处理24 h,用双荧光素酶报告基因检测系统分析外源性和内源性AR的转录活性。(3)取对数生长期LNCa P细胞,以K_(2)CrO_(4)0.5,2.0和8.0μmol·L^(-1)处理24 h,用逆转录PCR法检测AR和SENP家族分子SENP1,SENP2,SENP3,SENP5,SENP6,SENP7和SENP8 m RNA水平,Western印迹法检测AR,SENP1和SENP3蛋白水平。结果(1)与细胞对照组相比,K_(2)CrO_(4)浓度≥1.0μmol·L^(-1)时293T细胞存活率显著降低(P<0.01),K_(2)CrO_(4)浓度≥2.0μmol·L^(-1)时LNCa P细胞存活率明显降低(P<0.01)。(2)与细胞对照组相比,K_(2)CrO_(4)浓度≥1.0μmol·L^(-1)时293T细胞中外源性AR的转录活性显著增强(P<0.05),K_(2)CrO_(4)4.0和8.0μmol·L^(-1)明显增强LNCa P细胞中内源性AR转录活性(P<0.05)。(3)与细胞对照组相比,K_(2)CrO_(4)0.5,2.0和8.0μmol·L^(-1)均不改变LNCa P细胞中AR m RNA和蛋白水平,而K_(2)CrO_(4)2.0和8.0μmol·L^(-1)组SENP1 m RNA水平显著增加(P<0.05),K_(2)CrO_(4)0.5,2.0和8.0μmol·L^(-1)组SENP3 m RNA水平显著增加(P<0.05)。与细胞对照组相比,K_(2)CrO_(4)8.0μmol·L^(-1)组SENP1蛋白水平显著增加(P<0.01),K_(2)CrO_(4)2.0和8.0μmol·L^(-1)组SENP3蛋白水平显著增加(P<0.05)。结论K_(2)CrO_(4)可能通过上调SENP1和SENP3表达而增强AR转录活性。

OBJECTIVE To explore the effects of ChromiumⅥ(CrⅥ)on the transcription activity of androgen receptor(AR)and whether its underlying mechanism is related to the SUMO-specific protease(SENP)family of molecules.METHODS 293T cells were incubated with K_(2)CrO_(4)at 0.25,0.5,1.0,2.0,4.0,8.0 and 16.0μmol·L^(-1),respectively,for 24 h.LNCa P cells were incubated with K_(2)CrO_(4)at 0.25,0.5,1.0,2.0,4.0,10.0 and 20.0μmol·L^(-1),respectively,for 24 h.Cell viability was detected by methyl thiazolyl tetrazolium(MTT)assay and 50%inhibitive concentration(IC_(50))of CrⅥwas calculated.To detect the effects of CrⅥon the transcriptional activity of the exogenous AR,293T cells were co-transfected with AR and androgen response element(ARE)-luc reporter plasmids before being treated with K_(2)CrO_(4)at 0.25,0.5,1.0,2.0,4.0,8.0 and 16.0μmol·L^(-1),respectively,for 24 h.To detect the effects of Cr Ⅵ on the transcriptional activity of the endogenous AR,LNCa P cells were transfected with ARE-luc reporter plasmids,before being treated with K_(2)CrO_(4)at 0.25,0.5,1.0,2.0,4.0,8.0 and 16.0μmol·L^(-1),respectively,for 24 h.The transcriptional activity of AR was analyzed by the dual-luciferase reporter assay system.LNCa P cells in the exponential phase were incubated with K_(2)CrO_(4)at 0.5,2.0 and 8.0μmol·L^(-1),respectively,for 24 h.The m RNA levels of AR,SUMO-specific protease(SENP)1,SENP2,SENP3,SENP5,SENP6,SENP7 and SENP8 were measured by RT-PCR.The protein levels of AR,SENP1 and SENP3 were detected by Western blot.RESULTS Compared with the vehicle group,K_(2)CrO_(4)at the concentration of 1.0μmol·L^(-1)or above significantly decreased cell survival in 293T cells(P<0.01),and K_(2)CrO_(4)at the concentration of 2.0μmol·L^(-1)or above significantly shortened cell survival in LNCa P cells(P<0.01).Compared with the vehicle group,K_(2)CrO_(4)at the concentration of 1.0μmol·L^(-1)or above enhanced the transcriptional activity of the exogenous AR in 293T cells(P<0.05),and K_(2)CrO_(4)at 4.0and 8.0μmol·L^(-1)enhanced the transcriptional activity of the endogenous AR in LNCa P cells(P<0.05).Compared with the vehicle group,K_(2)CrO_(4)at 0.5,2.0 and 8.0μmol·L^(-1)had no effect on m RNA or protein levels of AR in LNCa P cells.Compared with the vehicle group,K_(2)CrO_(4)at 2.0 and 8.0μmol·L^(-1)increased SENP1 m RNA levels(P<0.05),and K_(2)CrO_(4)at 0.5,2.0 and 8.0μmol·L^(-1)elevated SENP3 m RNA levels(P<0.05)in LNCa P cells.Compared with the vehicle group,K_(2)CrO_(4)at 8.0μmol·L^(-1)increased SENP1protein levels(P<0.01),while K_(2)CrO_(4)at 2.0 and 8.0μmol·L^(-1)elevated SENP3 protein levels(P<0.05)in LNCa P cells.CONCLUSION These results indicate that CrⅥcan enhance transcriptional activity of AR by increasing the levels of SENP1 and SENP3.