RNAi抑制Akt2基因表达对卵巢癌细胞系A2780放射敏感性的影响  

Influence of Akt2 gene repression by RNA interference on the radiosensitivity of human ovarian cancer cell line A2780

作　　者：孙冬岩[1] 翁丹卉[1] 宋晓红[1] 夏曦[1] 卢运萍[1] 马丁[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：《中国癌症杂志》2008年第2期96-100,共5页China Oncology

基　　金：国家973重点基础研究发展计划资助项目(No:2002CB513107);国家自然科学基金资助项目(No:30571950)

摘　　要：背景与目的:许多研究已经表明蛋白激酶B(PI3K/Akt)在肿瘤细胞恶性增殖及肿瘤对放化疗的拮抗中起着重要作用,Akt2是Akt家族成员之一。本研究应用RNAi(RNA interference,RNAi)技术抑制Akt2基因的表达,观察其对肿瘤细胞生长和放射敏感性的影响。方法:构建Akt2基因的短发卡状RNA质粒转染人卵巢癌细胞A2780,同时以转染空载体和未转染组作为对照,3组细胞命名为pAkt2-shRNA/A2780,pEGFP/A2780和A2780)运用RT-PCR、蛋白质免疫印迹方法比较转染前后Akt2表达的差异;四甲基偶氮唑蓝实验绘制细胞生长曲线;克隆形成实验观察细胞的放射敏感性变化,以克隆形成率表示;6MV X线10Gy照射后48h收集细胞,流式细胞仪(FACS)分析细胞凋亡情况。结果:与对照相比,shRNA可抑制Akt2mRNA转录和蛋白的表达,差异显著(P<0.05);pAkt2-shRNA/A2780细胞生长明显慢于pEGFP/A2780和A2780细胞;pAkt2-shRNA/A2780细胞克隆形成率明显低于pEGFP/A2780细胞和A2780细胞(P<0.05);pAkt2-shRNA/A2780,pEGFP/A2780和A2780细胞凋亡率分别为(78.3±2.2)%、(41.2±1.8)%和(40.4±2.0)%,差异有显著性(P<0.05)。结论:转染针对Akt2基因shRNA真核表达载体,抑制卵巢癌细胞中Akt2基因表达,能抑制细胞增殖,促进细胞凋亡,增强其对放射治疗的敏感性。Background and purpose： Protein kinase B（ PI3K/ Akt）plays an important role in malignant proliferation, drug resistance and radioresistance of tumor cell, Akt2 is one of the Akt family members. This study was designed to explore the influence of Akt2 gene repression by RNAi on the proliferation and radiosensitivity of human ovarian cancer cell line A2780. Methods： Vectors containing shRNA targeting Akt2 gene were constructed and transfected into human ovarian carcinoma cell lines A2780, The RT-PCR and Western blot were used to compare the efficiency of gene silencing; The proliferation of cells was measured by methyl thiazolyl tetrazolium（MTT） ; The radiosensitivity of ovarian carcinoma was evaluated by Clone formation array, the cell apoptosis was detected by flow cytometry after 48 hours incubation at 10 Gy irradiation. Empty plasmid-transfected A2780 and normal A2780 were used as control. Results： Two stable transfected cell lines pAkt2-shRNA/A2780 and pEGFP/A2780 were screened from the transfected A2780. Western blot analyses and RT-PCR indicated that the expression of Akt2 was suppressed by the RNAi. The proliferation rate of pAkt2-shRNA/A2780 was obviously slower than control groups; the radiosensitivity of A2780 cells was enhanced after Akt2 gene repression, the apoptosis rate of pAkt2-shRNA/A2780, pEGFP/A2780 and A2780 cells were （78.3 ± 2.2） %, （41.2 ± 1.8） % 和 （40.4 ± 2.0） %, the difference was statistically significant（ P 〈 0.05）. Conclusions： Akt2 gene repression by RNAi can induce cell apoptosis, inhibit cell proliferation and obviously increase the radiosensitivity in human ovarian cell lines.

关 键 词：卵巢肿瘤 RNA干扰 放射敏感性 细胞凋亡 AKT2 

分 类 号：R737.33[医药卫生—肿瘤]