雄激素受体及共调节因子在睾酮调节绵羊附睾GPX5表达中的作用分析  

Regulation of GPX5 expression in sheep epididymis by androgen receptor and coregulators

作　　者：栾兆进 赵勇超 许娇霞 刘永斌 张家新[1] LUAN Zhaojin;ZHAO Yongchao;XU Jiaoxia;LIU Yongbin;ZHANG Jiaxin(Inner Mongolia Key Laboratory of Animal Genetics,Breeding and Reproduction,College of Animal Science,Inner Mongolia Agricultural University,Hohhot,Inner Mongolia 010018,China;College of basic medicine and forensic medicine,Baotou Medical College,Baotou,Inner Mongolia 010040,China;Academy of agriculture and animal husbandry of Inner Mongolia Autonomous Region,Hohhot,Inner Mongolia 010018,China)

机构地区：[1]内蒙古农业大学动物科学学院/内蒙古自治区动物遗传育种与繁殖重点实验室,呼和浩特010018 [2]包头医学院基础医学与法医学院,包头014040 [3]内蒙古自治区农牧业科学院,呼和浩特010018

出　　处：《中国农业大学学报》2023年第3期94-108,共15页Journal of China Agricultural University

基　　金：内蒙古自治区科技重大专项(2020ZD0003);内蒙古自治区自然科学基金项目(2022QN08016)。

摘　　要：为探究雄激素受体(Androgen receptor,AR)及共调节因子对绵羊附睾上皮细胞(Epididymal epithelial cells,EECs)谷胱甘肽过氧化物酶5(Glutathione peroxidase 5,GPX5)的调节机制,本研究采用CCK-8检测睾酮对EECs增殖的影响;利用qRT-PCR、免疫荧光法和Western Blot法分别检测AR、GPX5、甾体激素受体共激活子1(Steroid receptor coactivator 1,SRC-1)、CREB结合蛋白(cAMP-response element-binding protein,CBP)、p300和核受体共抑制因子2(Nuclear receptor corepressor 2,NCOR2)的mRNA水平和蛋白表达情况,采用双荧光素酶报告基因测定干扰SRC-1或p300后EECs的荧光素酶活性。结果表明:1)与对照组相比,100 nmol/L睾酮组细胞中的GPX5 mRNA和蛋白表达量极显著升高(P<0.01),1000 nmol/L睾酮组细胞中的GPX5 mRNA和蛋白表达量差异不显著(P>0.05),但分别极显著(P<0.01)和显著(P<0.05)低于100 nmol/L睾酮组;100 nmol/L睾酮组细胞中的AR mRNA和蛋白表达量分别呈极显著(P<0.01)和显著(P<0.05)高于对照组,然而1000 nmol/L睾酮组细胞中的AR mRNA和蛋白的表达量显著低于对照组(P<0.05);1000 nmol/L睾酮组细胞中的CBP、p300、SRC-1蛋白表达极显著高于对照组(P<0.01),特别是核内的表达量明显增高。2)与对照组相比,siRNA-AR组细胞中的GPX5 mRNA和蛋白的表达量差异不显著(P>0.05),而SRC-1和p300蛋白表达量极显著升高(P<0.01)。pcDNA3.1-AR组GPX5 mRNA和蛋白表达量较对照组呈极显著(P<0.01)和显著(P<0.05)升高。3)siRNA-SRC-1和siRNA-p300组细胞中的GPX5 mRNA和蛋白表达量较对照组均显著降低(P<0.05),而AR的蛋白表达量与对照组差异不显著(P>0.05),AR的转录活性显著降低(P<0.05)。综上,睾酮对绵羊EECs GPX5、AR及其共调节因子的表达具有浓度依赖性的调节效应,睾酮通过AR及其共调节因子SRC-1和p300/CBP的协同调节GPX5表达。本研究为探究GPX5在绵羊附睾中的调节机制提供理论依据。The purpose of this study was to investigate the regulatory mechanism of androgen receptor(AR)and co-regulatory factors in the regulation of testosterone on glutathione peroxidase 5(GPX5)in sheep epididymal epithelial cells(EECs).CCK-8 was used to detect the effect of testosterone on the proliferation of EECs.The mRNA levels and protein expressions of AR,GPX5,steroid receptor coactivator 1(SRC-1),cAMP-response element-binding protein(CBP),P300 and Nuclear receptor corepressor 2(NCOR2)were detected by qRT-PCR,immunofluorescence and Western blot;The luciferase activity of EECs after siRNA interference with SRC-1 or P300 was measured by double luciferase reporter gene.The results showed that:1)Compared with the control group,the expression of GPX5 mRNA and protein in 100 nmol/L testosterone group was significantly increased(P<0.01),while the expression of GPX5 mRNA and protein in 1000 nmol/L testosterone group was not significantly different from that in the control group(P>0.05),but it was significantly(P<0.01)and significantly(P<0.05)lower than that in 100 nmol/L testosterone group.The expression of AR in the nucleus was significantly increased(P<0.05),and the expression of AR mRNA and protein were significantly increased(P<0.01)and significantly(P<0.05),respectively.The expression of AR mRNA and protein in 1000 nmol/L testosterone group was significantly lower than that in the control group(P<0.05),and the expression of CBP,P300 and SRC-1 protein in 1000 nmol/L testosterone group increased significantly(P<0.01),especially in the nucleus.2)There was no significant difference in the expression of GPX5 mRNA and protein between siRNA-AR group and control group(P>0.05),while the expression of SRC-1 and P300 protein was significantly higher(P<0.01).The expression of GPX5 mRNA and protein in pcDNA 3.1-AR group was significantly higher than that in the control group(P<0.01)and significantly(P<0.05).3)The expression of GPX5 mRNA and protein in siRNA-SRC-1 group or siRNA-p300 group was significantly lower than that in the

关 键 词：附睾上皮细胞 谷胱甘肽过氧化物酶5 睾酮 雄激素受体 共调节因子 

分 类 号：Q826.9[生物学]