雄激素受体辅调节因子BAP18在大鼠睾丸细胞中的表达及其意义

Expression of androgen receptor co-regulator BAP18 in testicular cells of rats and its significance

目的:观察溴区PHD结构域转录因子(BPTF)相关蛋白18(BAP18)在睾丸细胞中的表达及其在雄激素受体(AR)信号通路过程中发挥的作用,阐明BAP18调控AR信号通路的具体靶基因,探讨BAP18在睾丸细胞中发挥的生物学功能。方法:收集大鼠睾丸支持细胞R2C和TM3及睾丸间质细胞TM4和15P-1,实时荧光定量PCR(RT-qPCR)法检测各细胞中BAP18和AR mRNA表达水平,Western blotting法检测细胞中BAP18和AR蛋白表达水平,蛋白免疫共沉淀实验(co-IP)检测在睾丸R2C细胞中BAP18与AR之间的相互作用关系。在R2C细胞中采用带有FLAG标签的BAP18过表达质粒使BAP18过表达(BAP18过表达组)。荧光素酶双基因报告实验检测R2C细胞中荧光素酶活性。对照组采用PcDNA3空载质粒转染R2C细胞,RT-qPCR法检测BAP18过表达前后睾酮(T)和双氢睾酮(DHT)作用后R2C细胞中BAP18、AR、STAR、HSD3B1、CYP17A1和DDX25 mRNA水平,Western blotting法检测BAP18过表达前后T和DHT作用后R2C细胞中CYP17A1蛋白表达水平。结果:在睾丸间质细胞和支持细胞中均有BAP18表达,但AR主要在睾丸间质细胞中表达;在睾丸R2C细胞中BAP18与AR存在相互作用,且上调AR介导的基因转录。与对照组比较,在T和DHT作用下BAP18过表达组R2C细胞中CYP17A1 mRNA和蛋白表达水平升高(P<0.01)。在R2C细胞中同时外源转染定量BAP18表达质粒后,在DHT的作用下细胞中荧光素酶活性升高近3倍,而在T的作用下,细胞中荧光素酶活性升高1.5倍。过表达BAP18后,BAP18过表达组R2C细胞中AR、STAR、HSD3B1和DDX25 mRNA表达水平与过表达前比较差异无统计学意义(P>0.05),而CYP17A1 mRNA表达水平高于过表达前(P<0.05);T和DHT作用后,R2C细胞中CYP17A1 mRNA表达水平均高于T和DHT作用前(P<0.05)。在细胞中过表达BAP18后,T和DHT作用后细胞中CYP17A1蛋白表达水平明显高于T和DHT作用前(P<0.05),但T和DHT刺激前后细胞中DDX25蛋白表达水平比较差异无统计学意义(P>0.05)。结论:BAP18可以在睾丸间质细胞中与AR相互作用并调控AR信号通路重要靶基因CYP17A1,可能参与睾丸间质细胞的生理过程。

Objective:To observe the expression of bromodomin PHD domain transcription factor(BPTF)associated protein18(BAP18)in testicular cells of the rats and its role in the androgen receptor(AR)signaling pathway,and to clarify the specific target gene of BAP18 directly regulating in the AR signaling pathway,and explore the biological capacity of BAP18 in the testicular cells.Methods:The sertoli cells and leydig cells of the rats were collected.The expression levels of BAP18 and AR mRNA in the cells were detected by real time fluorescence quantitative PCR(RT-qPCR)method,the expression levels of BAP18 and AR proteins in the cells were detected by Western blotting method,and the interaction between BAP18 and AR in the testicular R2C cells was detected by protein immunoprecipitation assay(co-IP).The BAP18 over-expression plasmid with FLAG tag was used to over-express the BAP18 in the R2C cells(BAP18 over-expression group).Luciferase double gene report test was used to detect the luciferase activity in the cells.In control group,the pcDNA3 empty plasmid was used to transfect the R2C cells.The expression levels of BAP18,AR,STAR,HSD3B1,CYP17A1,and DDX25 mRNA in the R2C cells treated with testosterone(T)and dihydrotestosterone(DHT)before and after BAP18 over-expression were detected by RT-qPCR method.The expression levels of CYP17A1 protein in the R2C cells treated with T and DHT before and after BAP18 over-expression were detected by Western blotting method.Results:BAP18 was expressed in the leydig cells and sertoli cells,but AR was mainly expressed in the leydig cells;BAP18 interacted with AR and up-regulated the AR-mediated gene transcription in the testicular R2C cells.Compared with control group,after over-expression of BAP18 under the action of T and DHT,the mRNA and protein expression levels of CYP17A1 in the R2C cells in BAP18 over-expression group were increased(P<0.01).After exogenous transfection of quantitative BAP18 expression plasmid in R2C cells at the same time,the luciferase activity in R2C cells was increased nearly 3 times under the action of DHT,while the luciferase activity in R2C cells was increased 1.5 times under the action of T.After BAP18 over-expression of in the R2C cells,the expression levels of AR,STAR,HSD3B1,and DDX25 mRNA in the R2C cells in BAP18 over-expression group were not significantly different from those before BAP18 over-expression(P>0.05),while the expression level of CYP17A1 mRNA was higher than that before BAP18 over-expression(P<0.05);after T or DHT treatment,the expression levels of CYP17A1 mRNA in the R2C cells were higher than before T or DHT treatment(P<0.05).After BAP18 over-expression in the cells,the expression levels of CYP17A1 protein in the cells after T or DHT treatment were significantly higher than before T or DHT treatment(P<0.05),but there was no significant difference in the expression level of DDX25 protein before and after T or DHT treatment(P>0.05).Conclusion:BAP18 can interact with AR in the Leydig cells and regulate the important target gene CYP17A1 of AR signaling pathway,which may be involved in the physiological process of Leydig cells.