雄激素应答元件陷阱DNA抑制PSA基因启动子的作用并诱导LNCaP细胞凋亡

摘要

AIM: To observe the effect of exogenous androgen responsive element decoy on the promoter of prostate specific antigen (PSA) and the growth of LNCaP cells for searching the possibility of gene therapy for prostate cancer. METHODS: Firstly, pGL3 - PSA luciferase expression vector containing 640bp - promoter fragment of PSA gene was constructed. Then, a 23 -mer phosphorothioated ARE decoy based on the deduced ARE sequence at the promoter region of PSA gene was synthesized. pGL3 - PSA and ARE decoy DNA were cotransfected into PC3 - M cell by lipofectamineTM 2000. Through detecting the activity of luciferase, the effect of ARE decoy on the promoter of PSA was studied. Then the ARE decoy DNA was transfected into LNCaP cells. The effect of decoy DNA on the proliferation of LNCaP cells was examined by using MTT assay. The effects of apoptosis were detected by phase contrast microscopy, DNA agrose gel electrophoresis and flow cytometry. Meanwhile, the nuclear extract was prepared from LNCaP cells and DNA - protein interactions were examined by electrophoretic mobility shift assay (EMSA). RESULTS: The reporter assay showed that the aetivity of luciferase was significantly reduced in the ARE decoy - transfected cells, bnt not in the cells transfected with the control decoy. EMSA demonstrated specific binding of the ARE decoy to androgen receptor. The growth of LNCaP was remarkably inhibited and apoptotic morphological changes as well as DNA fragmentation were observed in the ARE decoy- transfected cells. The rate of apoptosis was 22.4% detected by FCM. CONCLUSION: The ARE decoy is capable of inhibiting the promoter of PSA gene and inducing the apoptosis in prostate cancer cells. It may become a potential therapeutic tool for prostate cancers.%目的:研究雄激素应答元件陷阱DNA(ARE decoy)对前列腺特异抗原(PSA)基因启动子的抑制作用及其对前列腺癌细胞LNCaP细胞生长活性的影响,为前列腺癌的基因治疗寻求新的策略.方法:联合运用报告基因和陷阱DNA策略,构建含PSA基因5'侧启动子区640 bp DNA的荧光素酶表达载体pGL3-PSA,ARE陷阱DNA共转染前列腺癌细胞株PC3-M.应用双荧光素酶测定系统,检测荧光素酶的表达活性.然后,应用2 mg/L AREdecoy转染LNCaP细胞,通过相差显微镜观察细胞超微结构变化,MTT比色法检测细胞生长活性,DNA ladder和流式细胞技术(FCM)检测细胞凋亡以研究ARE decoy DNA对前列腺癌细胞LNCaP细胞生长活性的影响.同时提取LN-CaP细胞核蛋白,应用电泳迁移率变动分析(EMSA)检测ARE decoy DNA与雄激素受体的特异结合.结果:ARE de-coy DNA显著抑制报告基因荧光素酶的表达,抑制率可达95%.EMSA显示ARE decoy DNA能特异与核蛋白中雄激素受体结合.LNCaP细胞转染ARE decoy DNA后,镜下观察部分细胞出现凋亡形态学的改变,细胞体外生长受到抑制,染色体DNA凝胶电泳可见明显梯形条带.转染48h的凋亡率为22.4%.结论:实验表明ARE decoy DNA能竞争结合雄激素受体(AR),阻断AR的作用而诱导LNCaP细胞凋亡,有可能为前列腺肿瘤的治疗提供新的策略.