Decoy核酸在前列腺癌LNCaP细胞株中的作用及其机制

The role of decoy nuclear acid in LNCaP cells

目的 观察雄激素受体反应元件陷阱核酸（ARE decoy DNA）在LNCaP细胞中的致凋亡作用及下游信号通路.方法 根据ARE-Ⅰ、ARE-Ⅱ人工合成decoy核酸,进行部分和完全硫代修饰,应用凝胶阻抑分析方法检测4种decoy核酸与雄激素受体（AR）特异性结合.选择特异性结合最强decoy核酸通过LipofectinTM 2000转染细胞,MTS法检测转染后细胞增殖能力,共聚焦显微镜观察细胞形态学改变,Western blot检测细胞内蛋白表达.结果 ARE-Ⅱdecoy转染后MTS检测细胞A值0.500±0.013（48 h）,与对照decoy转染组0.750±0.055（48 h）比较差异有统计学意义（P＜0.01）.细胞核皱缩,核染色质分块,DNA电泳出现阶梯状ladder,证明细胞发生凋亡.Western blot检测发现细胞内p-Akt表达明显降低（P＜0.05）,Akt信号通路被抑制,促进了细胞凋亡.结论 ARE-Ⅱdecoy核酸明显抑制前列腺癌LNCaP细胞生长,促进细胞凋亡,改变细胞内信号通路.

Objective To investigate the role of ＂androgen responsive element （ARE） decoy＂ in LNCaP cells and its downstream signal pathway. Methods Four kinds of decoy with DNA-protein interactions were detected by gel shift assay （ EMSA）. Then LNCaP cells were transfected with the ARE decoy by LipofectinTM 2000.Cell viability was detected by MTS assay, and apoptosis was examined by DNA fragmentation. Confocal microscopy was used to observe cell morphology changes, and Western blotting analysis was used to detect p-Akt protein changes. Results MTS assay showed cellular proliferation was inhibited significantly by ARE- Ⅲdecoy with total sulfurized A value being 0.500 ±0.013 （48 h） as compared with control decoy [0.750 ±0.055 （48 h） , （P 〈0.01） ]. Morphologcally, apoptosis occurred in transfected cells. Western blotting analysis revealed a significant reduction of p-Akt protein （ P 〈 0.05）. Conclusion Total sulfurized ARE- D decoy had an antiandrogen effect and induced apoptosis in LNCaP cells by reducing the p-Akt protein expression.