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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。
This study investigated the effects of triptolide (TP) on the expression of androgen receptor (AR) in human prostate cancer LNCa P cells. The mRNA and protein expression of AR in LNCa P cells treated with TP and the changes of AR protein expression in LNCa P cells treated with TP and NF-κB inhibitor were detected by real-time PCR and Western blot. A series of pGL3-AR promoter reporter gene vectors were constructed and transfected into LNCa P cells to detect the regulatory effect of TP on AR transcription of LNCa P cells. Western blot was used to detect the upstream protein that may play a regulatory role. The results showed that the expression of m RNA and protein of AR in LNCa P cells decreased with the increase of drug concentration after 48 h of treatment with TP, and the expression of AR1 gene, PART1 and prostate specific antigen (PSA) Was also inhibited. A series of pGL3-AR promoter reporter vectors were successfully constructed. Sequencing results showed that the inserted sequence was correct and had no base mutation. After being transfected into LNCa P cells, the dual luciferase reporter gene system was verified to be active The results of promoter reporter assay showed that TP down-regulated the expression of AR at the transcriptional level and down-regulated the expression of PI3K / AKT / NF-κB pathway proteins related to AR promoter activity. Indicating that TP may down-regulate the transcriptional activity of AR in human prostate cancer LNCa P cells through the PI3K / AKT / NF-κB pathway.