CAJ | 학술논문

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).6MVX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Dq值均减小,放射增敏比(SERD0)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.
목적 이용RNA간우기술억제방사항거인비인암세포계CNE-2R중고표체적c-jun기인,연구기대CNE-2R세포방사민감성적영향급기잠재궤제.방법 설계합성3조특이성침대c-jun기인적siRNA,구건만병독vshRNA재체,감염CNE-2R세포,채용RT-PCR화Western blot방법검측각조대목적기인적억제효과;병통과극륭형성실험측정기방사민감성,CCK-8실험검측세포적존활능력,류식세포술검측세포조망솔.결과 중조만병독c-jun-RNAi-LV2능명현하조CNE-2R세포중c-jun적mRNA화단백수평적표체(F =217.20、42.07,P＜0.05).6MVX사선연합c-jun-RNAi-LV2조재0、2、4、6화8 Gy조사후세포적흡광도치균현저저우미전염조화음성대조조(F=42.70 ～ 200.67,P＜0.05).극륭형성실험현시,c-jun-RNAi-LV2조여미전염조급음성대조조상비,SF2、D0화Dq치균감소,방사증민비(SERD0)위1.41.세포조망실험결과현시,미경조사적c-jun-RNAi-LV2조여연합2 Gy조사적조망솔분별위(20.93±1.99)％화(38.17±0.83)％,균고우미전염조화음성대조조적조망솔[0 Gy:(10.97±0.70)％화(20.43±0.25)％;2 Gy:(10.80±1.25)％화(19.53±1.50)％;F=50.54、330.14,P＜0.05].결론 RNA간우기술가유효억제방사항거인비인암세포계CNE-2R중고표체적c-jun기인,사세포방사민감성증고,기작용가능여c-jun기인적하조사세포증식능력감약,세포조망증다유관.
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 D0was 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.