短发卡状RNA抑制AKT2基因表达增强卵巢癌细胞对紫杉醇敏感性的研究  

Induced apoptosis of ovarian carcinoma cells by using hairpin siRNA interfering AKT2 kinase expression

作　　者：翁丹卉[1] 邢辉[1] 邓友星[1] 梁逢奇[1] 黄磊[1] 李芳[1] 马丁[1] 卢运萍[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院妇产科,湖北武汉430030

出　　处：《中华肿瘤防治杂志》2007年第14期1041-1045,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(30571950);国家973重点基础科学基金(2002CB513107)

摘　　要：目的:构建AKT2基因特异性短发卡状RNA(shRNA)真核表达载体,观察RNA干扰(RNAi)技术,对人卵巢癌A2780和SKOV3细胞中AKT2蛋白激酶表达的抑制作用,并探讨其促进细胞凋亡的机制。方法:运用基因工程技术,设计合成针对AKT2 mRNA的特异性shRNA,构建真核表达载体pAKT2-shRNA。将pAKT2-shRNA经脂质体(LipofectamineTM2000)包裹转染人卵巢癌A2780和SKOV3细胞,荧光显微镜观察细胞内DsRed红色荧光蛋白的表达,计算转染效率,半定量RT-PCR(Semi-quantitativeRT-PCR)、蛋白质免疫印迹(Western blot)检测AKT2的表达及干扰效率,流式细胞学检测法(FACS)检测细胞凋亡率,比较抑制AKT2基因前后卵巢癌细胞抵抗凋亡能力。结果:成功构建了pAKT2-shRNA载体。A2780和SKOV3细胞转染pAKT2-shRNA后,荧光显微镜下观察转染效率约40%;Semi-quantitativeRT-PCR和Western blot检测结果示,转染后AKT2 mRNA和蛋白表达均显著降低。FACS检测结果显示,转染pAKT2-shR-NA后,紫杉醇25nmol/L处理细胞12h,与空白对照组和阴性对照组相比,细胞凋亡率显著增加,P<0.05。结论:构建的pAKT2-shRNA真核表达载体能有效抑制AKT2基因表达;运用RNAi技术,降低卵巢癌细胞中AKT2基因表达水平,能增强肿瘤细胞对化疗药物的敏感性,促进肿瘤细胞凋亡。OBJECTIVE： To construct the short hairpin RNA （shRNA） eukaryotic expression vector specific to human AKT2 gene （pAKT2-shRNA） and investigate its silence effect on AKT2 kinase and the mechanism of the induced cell apoptosis. METHODS： shRNA targeting AKT2 mRNA was designed and synthesized, and pAKT2-shRNA eukaryotic expression vector was constructed. LipofectamineTM 2000 method was used to transfect pAKT2-shRNA into human ovarian carcinoma cell lines A2780 and SKOV3. The efficiency of transfection was evaluated by immunofluorescence microscopy, and the expression change of AKT2 was examined by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR） and Western blot. The induced apoptosis by pAKT2-shRNA was analyzed by FACS. RESULTS： pAKT2-shRNA plasmid was successfully constructed. pAKT2-shRNA was transfectd by A2780 and SKOV3 and the transfection efficiency was 400//00. RT-PCR and wed blot recalt showed that AKT2 expressions in A2780 and SKOV3 cells were obviously knocked down by pAKT2-shRNA. FACS analyses showed that, compared with the control groups, pAKT2-shRNA promoted inducing apoptosis in A2780 and SKOV3 cells after being processed with TAX （25 nmol/L） for 12 hours （P〈 0. 05 ）. CONCLUSIONS： pAKT2-shRNA significantly knocks down AKT2 expression at mRNA and protein level and promots to inducing apoptosis in ovarian carcinoma cells. The successful application of pAKT2-shRNA in ovarian carcinoma cells may enhance the therapeutic effect of chemotherapeutics.

关 键 词：卵巢肿瘤 RNA干扰 基因表达 细胞凋亡 

分 类 号：R737.31[医药卫生—肿瘤]