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目的:研究雄激素应答元件陷阱DNA(AREdecoy)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640bpDNA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2mg/LAREdecoy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,DNAladder和流式细胞技术(FCM)检测细胞凋亡以研究AREdecoyDNA对前列腺癌细胞LNCaP细胞生长活性的影响。同时提取LNCaP细胞核蛋白,应用电泳迁移率变动分析(EMSA)检测AREdecoyDNA与雄激素受体的特异结合。结果:AREdecoyDNA显著抑制报告基因荧光素酶的表达,抑制率可达95%。EMSA显示AREdecoyDNA能特异与核蛋白中雄激素受体结合。LNCaP细胞转染AREdecoyDNA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AREdecoyDNA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。
OBJECTIVE: To study the inhibitory effect of androgen receptor element (AREdecoy) on the promoter of prostate specific antigen (PSA) gene and its effect on the growth of prostate cancer cell line LNCaP in order to find a new strategy for gene therapy of prostate cancer. METHODS: A luciferase expression vector pGL3-PSA containing a 640 bp DNA sequence encoding the 5 ’flanking region of PSA gene was constructed by using a combination of reporter gene and trap DNA strategy. The ARE DNA was co-transfected into PC3-M prostate cancer cell line. Dual luciferase assay system was used to detect luciferase activity. Then, LNCaP cells were transfected with 2 mg / LAREdecoy, the changes of cell ultrastructure were observed by phase contrast microscopy, the cell growth activity was detected by MTT colorimetric assay, apoptosis was detected by DNAladder and flow cytometry (FCM) to study the effect of AREdecoyDNA on prostate cancer cells LNCaP cell growth activity. At the same time, LNCaP nuclear protein was extracted, and the specific binding of AREdecoyDNA to androgen receptor was detected by electrophoretic mobility shift assay (EMSA). Results: AREdecoyDNA significantly inhibited the luciferase reporter gene expression, the inhibition rate of up to 95%. EMSA showed that AREdecoyDNA specifically binds to the androgen receptor in nucleoprotein. After transfection of AREdecoyDNA in LNCaP cells, morphological changes of apoptotic cells were found in some cells under microscope. The growth of cells was inhibited in vitro. The obvious trapezoidal bands were observed on chromosome DNA gel electrophoresis. The apoptosis rate of transfected 48h was 22.4%. Conclusion: The results showed that AREdecoyDNA can competitively bind with androgen receptor (AR) and block the action of AR to induce apoptosis of LNCaP cells, which may provide a new strategy for the treatment of prostate tumors.