雷公藤内酯醇对人LNCaP细胞雄激素受体表达的调控及其机制

Effects of triptolide on the expression of androgen receptor in human prostate LNCaP cells and its mechanism of action

本文研究了雷公藤内酯醇(triptolide,TP)对人前列腺癌LNCa P细胞内雄激素受体(androgen receptor,AR)表达影响的作用机制。采用实时定量PCR和Western blot方法检测TP处理后LNCa P细胞中AR的m RNA和蛋白表达的变化,以及使用TP和NF-κB抑制剂共处理后LNCa P细胞AR蛋白表达的变化;采用RF克隆法构建系列p GL3-AR启动子报告基因载体并转染至LNCa P细胞,检测TP对LNCa P细胞AR转录的调节作用;采用Western blot检测可能起调节作用的上游蛋白。结果表明,TP处理LNCa P细胞48 h后,LNCa P细胞AR的m RNA和蛋白的表达随着药物浓度增加而下降,同时AR靶基因PART1及前列腺特异性抗原(prostate specific antigen,PSA)的表达也受到抑制;成功构建系列p GL3-AR启动子报告基因载体,测序结果表明,插入的目的序列正确且无碱基突变,转染LNCa P细胞后双荧光素酶报告基因检测系统证实其具有启动子活性;启动子报告基因实验结果表明,TP在转录水平下调AR的表达;与AR启动子活性相关的PI3K/AKT/NF-κB通路蛋白表达下调。说明TP可能通过PI3K/AKT/NF-κB通路下调人前列腺癌LNCa P细胞AR的转录活性。

To study the regulation of androgen receptor （AR） expression in human prostate cancer LNCaP cells by triptolide （TP） and the possible mechanism, by using qRT-PCR and Western blot, the AR mRNA and protein levels in TP treated LNCaP cells were detected, and the AR protein level in TP and NF-κB inhibitor treated LNCaP cells was also detected; a series of pGL3-AR promoter reporter gene vectors were built using restriction-free cloning method, and the vectors were employed to investigate the effects of TP on the transcriptional activity of AR promoter in LNCaP cells; the upstream proteins which may play regulatory roles were detected using western blot assay. After treated LNCaP cells with TP for 48 h, AR mRNA and protein expressions decreased with increasing TP concentration. The expression of AR target gene PART1 and prostate specific antigen （PSA） was also downregulated by TP treatment; a series of pGL3-AR promoter reporter vectors were constructed and validated by sequencing and luciferase activity; the results of dual luciferase reporter assay showed that TP downregulated AR at the transcriptional level; PI3K/AKT/NF-κB pathway which isassociated with AR promoter activity was drowregulated by TP. In conclusion, our results demonstrated that the transcriptional activity of AR in LNCAP cells was downregulated by TP, and PI3K/AKT/NF-κB pathway may be involved in the regulation mechanism.