RNA干扰抑制c-jun基因表达对放射抗拒人鼻咽癌细胞系CNE-2R放射敏感性的影响

目的 利用RNA干扰技术抑制放射抗拒人鼻咽癌细胞系CNE-2R中高表达的c-jun基因,研究其对CNE-2R细胞放射敏感性的影响及其潜在机制.方法 设计合成3组特异性针对c-jun基因的siRNA,构建慢病毒vshRNA载体,感染CNE-2R细胞,采用RT-PCR和Western blot方法检测各组对目的基因的抑制效果;并通过克隆形成实验测定其放射敏感性,CCK-8实验检测细胞的存活能力,流式细胞术检测细胞凋亡率.结果 重组慢病毒c-jun-RNAi-LV2能明显下调CNE-2R细胞中c-jun的mRNA和蛋白水平的表达(F=217.20、42.07, P<0.05).6 MV X射线联合c-jun-RNAi-LV2组在0、2、4、6和8 Gy照射后细胞的吸光度值均显著低于未转染组和阴性对照组(F=42.70~200.67, P<0.05).克隆形成实验显示,c-jun-RNAi-LV2组与未转染组及阴性对照组相比,SF₂、D₀和D_q值均减小,放射增敏比(SER_D0)为1.41.细胞凋亡实验结果显示,未经照射的c-jun-RNAi-LV2组与联合2 Gy照射的凋亡率分别为(20.93±1.99)%和(38.17±0.83)%,均高于未转染组和阴性对照组的凋亡率0 Gy:(10.97±0.70)%和(20.43±0.25)%;2 Gy:(10.80±1.25)%和(19.53±1.50)%;F=50.54、330.14, P<0.05].结论 RNA干扰技术可有效抑制放射抗拒人鼻咽癌细胞系CNE-2R中高表达的c-jun基因,使细胞放射敏感性增高,其作用可能与c-jun基因的下调使细胞增殖能力减弱,细胞凋亡增多有关.

Effect of c-jun gene RNAi on the radiosensitivity of radioresistant human nasopharyngeal carcinoma cell line CNE-2R

Objective To investigate the effect of c-jun RNAi on the radiosensitivity of radioresistant human nasopharyngeal carcinoma cell line CNE-2R and the possible mechanisms. Methods Three siRNA sequences specific for c-jun gene were designed, and lentvirus vector with c-jun shRNA was constructed and infected into CNE-2R. The gene silencing efficiency of these recombinants was detected by RT-PCR and Western blot. Radiosensitivity, cell proliferation and apoptosis were measured using colony formation assay, CCK-8 assay and flow cytometry respectively. Results Recombinant lentiviral c-jun-RNAi-LV2 obviously inhibited c-jun expression at mRNA and protein level (F=217.20, 42.07, P<0.05). The cell proliferation of combination group (c-jun-RNAi-LV2 plus 0, 2, 4, 6, 8 Gy of 6 MV X-rays) was significantly lower than that of the other two groups (F=42.70-200.67, P<0.05). The cloning formation of c-jun-RNAi-LV2 group was significantly lower than that of non-transfected group and NC-transfected group and its sensitizing enhance rate (SER) at D₀ was 1.41. The apoptosis rates of 0, 2 Gy X-ray radiation in c-jun-RNAi-LV2 group, non-transfected group and NC-transfected group were(20.93±1.99)%, (38.17±0.83)%;(10.97±0.70)%, (20.43±0.25)%;(10.80±1.25)% and (19.53±1.50)% (F=50.54, 330.14, P<0.05), respectively. Conclusions RNA interference inhibited the c-jun expression and enhanced the radiation sensitivity of human nasopharyngeal carcinoma cell line CNE-2R, perhaps due to that the c-jun-RNAi-LV2 mediated c-jun down-regulation inhibited cell proliferation and promoted cell apoptosis.