shRNA沉默MRE11表达对肝癌耐药细胞BEL7402/5-FUDNA损伤修复的影响  

ShRNA-mediated MRE11 gene silencing inhibits DNA repair in multidrug-resistant hepatocellular carcinoma cell line BEL7402/5-FU

作　　者：范芳[1] 耿磊[1] 李大玉[1] 李长福[1] 

机构地区：[1]遵义医学院生化教研室,贵州省遵义市563099

出　　处：《世界华人消化杂志》2013年第29期3053-3058,共6页World Chinese Journal of Digestology

基　　金：贵州省科技基金资助项目;No.[2009]3066;遵义市红花岗区科技基金资助项目;No.(2009)18~~

摘　　要：目的:观察shMRE11沉默MRE11基因对肝癌耐药细胞Bel7402/5-FU DNA损伤修复功能的影响.方法:采用阳离子脂质体法将shMRE11干扰质粒转染BEL7402/5-FU细胞,Real-time PCR及Western blot检测沉默效率;Western blot检测细胞-H2AX蛋白表达;EdU法检测细胞DNA合成;MTT法检测细胞增殖情况.结果:Real-time PCR及Western blot检测结果显示MRE11 mRNA及蛋白水平的沉默效率分别为78.0%、56.1%;Western blot检测-H2AX表达结果显示shMRE11实验组(1.04±0.056)较对照组(0.847±0.025)增加(P<0.05,t=10.78);EdU检测结果显示shMRE11实验组DNA合成率(38.819%±2.607%)较对照组(49.814%±1.227%)降低(P<0.05,t=-8.87);MTT细胞增殖检测结果显示转染72 h后shMRE11实验组(0.58±0.08)与对照组(0.87±0.09)相比细胞增殖速度减慢(P<0.05,t=-50.2).结论:shMRE11干扰质粒能够有效抑制B E L7402/5-F U细胞中M R E11表达,使细胞DNA损伤修复能力减弱、抑制细胞增殖.AIM： To investigate the effect of shRNA-mediated MRE11 gene silencing on cell proliferation and apoptosis in multidrug-resistant hepatocellular carcinoma cell line BEL7402/5-FU. METHODS： A shRNA targeting the MRE11 gene was transiently transfected into BEL7402/5-FU cells using cathodolyte liposomes. Silencing efficiency was assessed using Real-time PCR and Western blot. The protein expression of γ-H2AX was detected by Western blot. Cellular DNA synthesis was detected by EdU. Cell proliferation was assessed by MTT assay. RESULTS： Real-time PCR and Western blot results showed that the efficiency of RNA interference for MRE11 was 78.0% at the mRNA level and 56.1% at the protein level. Western blot analysis demonstrated that γ-H2AX expression was significantly higher in the shMRE11 group than in the control group （1.04 ± 0.056 vs 0.847 ± 0.025, P 〈 0.05, t = 10.78）. DNA synthesis was significantly decreased in the shMRE11 group compared with the control group （38.819 ± 2.607 vs 49.814 ± 1.227, P 〈 0.05, t = -8.87）. The proliferation rate was significantly lower in the shMRE11 group than in the control group （0.58 ± 0.08 vs 0.87 ± 0.09, P 〈 0.05, t = -50.2）. CONCLUSION： ShRNA-mediated MRE11 gene silencing effectively inhibit DNA repair and proliferation of BEL7402/5-FU cells.

关 键 词：减数分裂重组蛋白11 RNA干扰 BEL7402 5-FU DNA损伤修复 

分 类 号：R735.7[医药卫生—肿瘤]