抑制NFBD1基因的表达对鼻咽癌CNE-1细胞放射敏感性的影响  

Effect of NFBD 1 gene silencing by RNA interference on radiosensitivity of nasopharyngeal carcinoma CNE-1 cells

作　　者：王志海[1] 曾泉[1] 陈弢[1] 廖奎[2] 卜友泉[3] 洪苏玲[1] 胡国华[1] 

机构地区：[1]重庆医科大学附属第一医院耳鼻咽喉科,重庆400016 [2]重庆医科大学附属第一医院肿瘤科,重庆400016 [3]重庆医科大学分子医学与肿瘤研究中心,生物化学与分子生物学教研室,重庆400016

出　　处：《肿瘤》2014年第2期101-107,共7页Tumor

基　　金：重庆市自然科学基金重点资助项目(编号:cstc2012jjB10015);重庆市卫生局2010年医学科研计划面上资助项目(编号:2010-2-020)

摘　　要：目的:通过短发夹RNA(short hairpin RNA,shRNA)干扰NFBD1(nuclear factor with BRCT domains protein 1)基因的表达,并探讨其对鼻咽癌CNE-1细胞放射敏感性的影响。方法:以慢病毒感染的方法将特异性针对NFBD 1基因的shRNA转入CNE-1细胞,并用嘌呤霉素筛选稳定表达的细胞;分别采用实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)及蛋白质印迹法检测NFBD1 mRNA和蛋白的表达;以克隆形成和FCM法检测沉默NFBD 1基因对CNE-1细胞放射敏感性的影响。结果:NFBD1-shRNA慢病毒载体感染后,CNE-1细胞中NFBD1 mRNA和蛋白表达水平明显降低。各组细胞经6 MeV电子线照射后,NFBD1-shRNA组细胞的凋亡率和G2/M期细胞所占的比例均较阴性对照组[negative control-shRNA(NC-shRNA)]明显增加(P<0.05),细胞存活分数较NC-shRNA组明显降低(P<0.05)。结论:采用慢病毒感染的方法靶向NFBD 1基因的RNA干扰可稳定沉默NFBD1的表达,从而增加鼻咽癌细胞对放射治疗的敏感性。Objective： To investigate the effect of nuclear factor with BRCT domains protein 1 （NFBD1） gene silencing by short hairpin RNA （shRNA） interference on the radiosensitivity of human nasopharyngeal carcinoma CNE-1 cells. Methods： The CNE-1 cells were transfected with NFBD1-shRNA by method of lentiviral infection, the stably transfected cells were selected by puromycin, and the mRNA and protein levels of NFBD1 in CNE-1 ce^s were detected by read-time fluorogenic quantitative-PCR （RFQ- PCR） and Western blotting, respectively. For the examination of the effect of NFBD1 depletion on the radiosensitivity of CNE-1 cells, the flow cytometry was used to calculate the apoptotic rate and cell cycle distribution, and the survival fraction of the cells was measured via colony formation assay. Results： After transfection with NFBDI-shRNA, the mRNA and protein levels of NFBD1 in CNE-1 cells were decreased obviously. The percentage of the cells in G2/M phase and the apoptotic rate of CNE-1 cells in NFBD1- shRNA group were increased after ionizing radiation （6 MeV） as compared with those of the negative control （NC）-shRNA group （P 〈 0.05）. The survival fraction of CNE-1 cells in NFBDI-shRNA group was significantly reduced after radiation as compared with that of the NC-shRNA group （P 〈 0.05）. Conclusion： Lentivirus-mediated shRNA targeting NFBD1 can steadily silence the expression of NFBD1 gene and enhance the radiosensitivitv of CNE-1 cells.

关 键 词：鼻咽肿瘤 RNA 小分子干扰 慢病毒感染 放射肿瘤学 

分 类 号：R739.63[医药卫生—肿瘤]