RNA干扰技术联合抑制AKT1和COX-2表达对U251胶质瘤细胞增殖的影响  

Suppressing effects of shRNA targeting AKT1 and COX-2 on proliferation of glioma cell U251 in vitro

作　　者：韩磊[1] 张安玲[1] 岳晓[1] 杨旸[1] 王广秀[1] 贾志凡[1] 浦佩玉[1] 康春生[1] 

机构地区：[1]天津医科大学总医院神经外科 天津市神经病学研究所神经肿瘤实验室,300052

出　　处：《中华神经外科杂志》2010年第12期1137-1140,共4页Chinese Journal of Neurosurgery

基　　金：基金项目：国家自然科学基金资助（30772231,30971136）;教育部新世纪优秀人才支持计划资助（NCET-07-0615）;天津市科技计划项目资助（09JCZDJC17600）;天津市卫生局科学基金资助（09KZ112）

摘　　要：目的 探讨应用RNA干扰（RNAi）技术抑制U251恶性胶质瘤细胞株AKT1和COX-2表达后,在体外对U251细胞增殖的抑制作用.方法 构建靶向AKT1和COX-2的RNAi重组腺病毒表达载体（rAd-A＋C）转染至U251细胞.应用Realtime PCR检测AKT1和COX-2 mRNA水平的变化;应用Western blot检测AKT1和COX-2蛋白水平的变化,并对肿瘤细胞中相关功能蛋白的表达进行分析;应用MTr法和流式细胞术评价肿瘤细胞的增殖活性.结果 rAd-A＋C可显著抑制U251细胞AKT1和COX-2的表达;与对照组和无义序列处理组（rAd-HK）比较,Western blot分析结果显示下游相关因子PCNA、Cyclin D1表达下降,而p53表达上调;细胞周期分析结果表明rAd-A＋C处理组进入S期的细胞数较对照组减少了7.0%～7.8%,出现G0/G1期阻滞;MTT法分析显示rAd-A＋C处理组细胞生长抑制率＞58%.结论 靶向AKT1和COX-2的RNAi技术可显著抑制U251细胞AKT1和COX-2表达,在体外对U251细胞增殖活性产生明显抑制作用.因此,RNAi重组腺病毒表达载体介导的基因治疗可成为胶质瘤靶向治疗的新策略.Objective To investigate the effects of knocking down AKT1 and COX-2 by RNAi on the proliferation activity of U251 glioma cells in vitro.Methods The recombinant adenovirus vector which contained RNAi expression construct of AKT1 and COX-2 （rAd-A ＋ C） was transfected into U251 cells.The silencing effects of RNAi on AKT1 and COX-2 expression were identified by realtime PCR and Western blot.Western blot was also undertaken to analyze the expression of some functional proteins.The proliferation activity of U251 cells transfected by rAd-A ＋ C was evaluated by MTT and flow cytometry.Result RAd-A ＋ C dramatically down-regulated the expression of AKT1 and COX-2 in U251 cells.As compared with parental and rAd-HK transfected cells,the protein expression of PCNA and CyclinDl was down-regulated in rAd-A ＋ C treated cells,meanwhile p53 was upregulated.Decreased AKT1 and COX-2 expression in rAd-A ＋ C transfected cells was accompanied by 7.0% to 7.8% decrease in U251 cells in S phase with G0/G1 phase arrest.Meanwhile,the proliferation activity was significantly decreased in rAd-A ＋ C transfected cells by the MTT assay.Conclusion RNAi targeting AKT1 and COX-2 could significantly down-regulate their expression in a sequence-specific manner,exerting proliferation inhibition effect on U251 cells in vitro.Consequently,the recombinant adenovirus vector mediated RNAi gene therapy may be a new strategy in targeting molecular therapy of gliomas.

关 键 词：神经胶质瘤 AKT1 COX-2 RNA干扰 增殖活性 

分 类 号：R686[医药卫生—骨科学]