EBV-miR-BART5-3p靶向p53对鼻咽癌细胞放射敏感性的影响  

Effect of EBV-miR-BART5-3p targeting p53 on radiosensitivity of nasopharyngeal carcinoma cells

作　　者：马庆华[1] 马菲[1] 李雪娟[1] 赵孟阳 MA Qinghua;MA Fei;LI Xuejuan;ZHAO Mengyang(Department of Otolaryngology,the Seventh People's Hospital of Zhengzhou,Henan Zhengzhou 450000,China)

机构地区：[1]郑州市第七人民医院耳鼻咽喉科,河南郑州450000

出　　处：《现代肿瘤医学》2021年第10期1655-1661,共7页Journal of Modern Oncology

基　　金：国家自然科学基金项目(编号:81702672)。

摘　　要：目的:研究爱泼斯坦-巴尔病毒(EBV)-miR-BART5-3p对鼻咽癌细胞放射敏感性的影响,并探讨其作用机制。方法:体外培养人EBV阳性鼻咽癌细胞(C666-1)和人鼻咽癌细胞(CNE-2Z),将CNE-2Z组细胞设置为正常组,C666-1细胞随机分为对照组、EBV-miR-BART5-3p NC组、EBV-miR-BART5-3p mimics组和EBV-miR-BART5-3p inhibitor组。用实时荧光定量PCR(RT-qPCR)法检测各组细胞及EBV阴性鼻咽癌患者和EBV阳性鼻咽癌患者癌组织中EBV-miR-BART5-3p表达情况,噻唑蓝(MTT)法检测各组细胞存活率,平板克隆实验评估各组放疗敏感性变化情况,膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)法检测各组细胞凋亡敏感性变化,蛋白免疫印迹分析法检测转染后各组细胞p53、Bcl-2相关X蛋白(Bax)、半胱天冬氨酸酶-3(caspase-3)和Bcl-2蛋白表达情况,双荧光素酶报告实验验证EBV-miR-BART5-3p与p53的靶向关系。结果:与EBV阴性组相比,EBV阳性组鼻咽癌组织中EBV-miR-BART5-3p表达水平显著升高(P<0.05)。与正常组相比,对照组EBV-miR-BART5-3p表达、存活率、克隆数量和Bcl-2蛋白表达水平显著升高(P<0.05),凋亡率、p53、Bax和caspase-3蛋白表达水平显著降低(P<0.05)。与对照组和EBV-miR-BART5-3p NC组相比,EBV-miR-BART5-3p mimics组EBV-miR-BART5-3p表达水平、存活率、克隆数量和Bcl-2蛋白表达水平显著升高(P<0.05),凋亡率、p53、Bax和caspase-3蛋白表达水平显著降低(P<0.05);与对照组和EBV-miR-BART5-3p NC组相比,EBV-miR-BART5-3p inhibitor组EBV-miR-BART5-3p表达水平、存活率、克隆数量和Bcl-2蛋白表达水平显著降低(P<0.05),凋亡率、p53、Bax和caspase-3蛋白表达水平显著升高(P<0.05)。双荧光素酶报告实验结果显示,与TP53-3'UTR-WT+EBV-miR-BART5-3p NC组比较,TP53-3'UTR-WT+EBV-miR-BART5-3p inhibitor组荧光素酶活性降低(P<0.05)。结论:下调EBV-miR-BART5-3p可能通过靶向促进p53蛋白表达,提高人EBV阳性鼻咽癌细胞的放射敏感性。Objective:To study the effect of Epstein-Barr virus(EBV)-miR-BART5-3p on the radiosensitivity of nasopharyngeal carcinoma cells and explore its mechanism.Methods:Human EBV positive nasopharyngeal carcinoma cells(C666-1)and human nasopharyngeal carcinoma cells(CNE-2Z)were cultured in vitro.CNE-2Z cells were set as normal group,and C666-1 cells were randomly divided into control group,EBV-miR-BART5-3p NC group,EBV-miR-BART5-3p mimics group and EBV-miR-BART5-3p inhibitor group.Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression of EBV-miR-BART5-3p in cells and tissues of nasopharyngeal carcinoma patients with EBV negative and EBV positive.Methyl thiazolyl tetrazolium(MTT)assay was used to detect the survival rate of cells in each group.The changes of radiosensitivity were evaluated by plate clone test.Annexin V-fluorescein isothiocyanate/propidium iodide(Annexin V-FITC/PI)assay was used to detect the apoptosis sensitivity.The protein expression of p53,Bcl-2 associated X protein(Bax),caspase-3 and Bcl-2 was detected by Western blot.In addition,double luciferase reporter assay was used to verify the targeting relationship between EBV-miR-BART5-3p and p53.Results:Compared with that in EBV negative group,the expression level of EBV-miR-BART5-3p was significantly higher in EBV positive group(P<0.05).Compared with those in the normal group,the expression of EBV-miR-BART5-3p,survival rate,clone number and Bcl-2 protein expression level were significantly higher in the control group(P<0.05),while the apoptosis rate,p53,Bax and caspase-3 protein expression level were significantly lower(P<0.05).Compared with those in the control group and EBV-miR-BART5-3p NC group,the expression level of EBV-miR-BART5-3p,survival rate,clone number and Bcl-2 protein expression level were significantly higher in EBV-miR-BART5-3p mimics group(P<0.05),while the apoptosis rate,p53,Bax and caspase-3 protein expression level were significantly lower(P<0.05),meanwhile,the expression level of EBV-miR-BART5-3p,survival ra

关 键 词：EBV-miR-BART5-3p P53 鼻咽癌细胞 放疗敏感性 

分 类 号：R739.6[医药卫生—肿瘤]