RNA干扰敲低Dicer对胶质瘤细胞恶性表型的影响  

The effect of silencing Dicer by RNA interference on malignant phenotype of glioma cells

作　　者：韩磊[1] 张安玲[1] 岳晓[1] 许鹏[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]天津市神经病学研究所神经肿瘤实验室,天津医科大学总医院神经外科300052

出　　处：《中华神经外科杂志》2011年第2期131-135,共5页Chinese Journal of Neurosurgery

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

摘　　要：目的 观察应用RNA干扰（RNAi）技术敲低Dicer酶后,在体外对U251人胶质瘤细胞生物学特征的影响.方法 构建针对Dicer基因的小分子干扰RNA重组腺病毒表达载体（rAd-Dicer）转染至U251细胞.应用RT-PCR检测Dicer mRNA水平的变化,应用免疫荧光和Western blot方法检测Dicer基因在蛋白水平的变化,并对肿瘤细胞中相关功能蛋白的表达进行分析比较.应用MTT法、流式细胞术和Transwell方法评价肿瘤细胞转染前后生物学行为的变化.结果 rAd-Dicer可显著抑制U251细胞Dicer基因的表达;与正常对照组（control）和阴性对照组（rAd-HK）比较,Western blot分析结果显示p-AKT,MMP2/9,PCNA,Cyclin a,VEGF,CD34功能蛋白在rAd-Dicer转染组细胞中表达明显上调;细胞周期结果表明rAd-Dicer转染组进入S期的细胞数增加了 14.1%～15.3%,MTT和Transwell实验结果显示rAd-Dicer转染组细胞增殖速率和体外侵袭能力显著增强.结论 敲低Dicer酶表达后,U251细胞表型具有更为恶性转化的倾向,由此初步推测全面抑制细胞microRNAs表达有可能促进肿瘤生成.Objective To investigate the effect of knocking down Dicer by RNAi on the biological characteristics of human glioma cells U251 in vitro.Methods The recombinant adenovirus expression vector which contained short hairpin RNA targeting Dicer （rAd-Dicer） was transfected into U251 cells.The silencing effect of RNAi on Dicer expression was identified by RT- PCR,immumofluorecence staining and Western blot.Western blot was also undertaken to analyze the expression of some functional proteins.The biological behavior changes of U251 cells trasfected by rAd- Dicer were evaluated by MTT,FCM and transwell assay.Methods rAd-Dicer dramatically down- regulated the expression of Dier in U251 cells.As compared with parental and rAd- HK transfected cells,the protein expression of p- AKT,MMP2/9,PCNA,Cyclin a,VEGF,CD34 were up- regulated in rAd-Dicer treated cells.Decreased dicer expression in rAd-Dicer transfected cells was accompanied by 14.1% to 15.3% increase in U251 cells in S phase.Meanwhile,the proliferation and invasive activity were significantly enhanced in rAd-Dicer transfected cells by the MTT and transwell assay.Conclusion Knockdown of dicer renders the U251 cells more malignant transformation.This preliminary finding suggests that global lowering expression of microRNAs may have an oncogenic role.

关 键 词：神经胶质瘤 microRNA DICER RNA干扰 生物学表型 

分 类 号：R739.4[医药卫生—肿瘤]