miR-18a促进自噬增强人鼻咽癌细胞株放疗敏感性的研究  

miR-18a enhances the radiosensitivity of nasopharyngeal carcinoma cells through inducing autophagy

作　　者：常利红[1] 姚周周 鲍宏伟 李越 陈晓红[1] 赖晓萍 黄子真[1] 张革化[1] Chang Lihong;Yao Zhouzhou;Bao Hongwei;Li Yue;Chen Xiaohong;Lai Xiaoping;Huang Zizhen;Zhang Gehua(Department of Otorhinolaryngology Head and Neck Surgery,the Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China)

机构地区：[1]中山大学附属第三医院耳鼻咽喉头颈外科,广州510630

出　　处：《中华耳鼻咽喉头颈外科杂志》2021年第7期736-745,共10页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：广东省科技计划项目(2017A020215073,2014A020212619);广东省自然科学基金博士启动项目(2015A030310073)。

摘　　要：目的:探索miR-18a过表达和抑制表达对人鼻咽癌细胞株CNE1和CNE2放疗敏感性的影响及可能的机制。方法:采用miR-18a过表达质粒miR-18a模拟物和抑制表达质粒miR-18a遏制物及空白对照质粒转染人鼻咽癌细胞株CNE1和CNE2细胞,实时荧光定量反转录聚合酶链反应(qRT-PCR)和蛋白免疫印迹法(WB)检测其对毛细血管扩张性共济失调症突变(ataxia telangiectasia mutated,ATM)基因和蛋白表达的影响。构建miR-18a过表达和抑制表达的人鼻咽癌细胞CNE1和CNE2细胞株。四甲基偶氮唑蓝(MTT)检测miR-18a过表达和抑制表达联合放疗处理前后对人鼻咽癌细胞株生长的影响,流式细胞术检测细胞凋亡和细胞周期,克隆形成实验检测细胞放疗敏感性,吖啶橙染色结合WB检测细胞自噬水平及自噬相关蛋白表达。采用SPSS 16.0软件进行统计学分析,2组间均数比较采用独立样本t检验。结果:100、200 nmol/L miR-18a模拟物转染CNE1和CNE2细胞48 h后,与对照组相比,ATM mRNA表达受到显著抑制(CNE1相对表达量为0.174±0.139和0.003±0.001,t值为9.939和19470.783;CNE2:相对表达量为0.024±0.008和0.019±0.012,t值为270.230和137.746,P值均<0.001),72 h后,ATM蛋白水平显著下降;100、200 nmol/L miR-18a遏制物转染48 h后,CNE1细胞中ATM转录显著增加(CNE1相对表达量为9.419±2.495和2.500±1.063,t值为-4.427和-41.241,P值均<0.05);CNE2细胞中ATM转录亦显著增加(相对表达量为7.210±0.171和115.875±15.805,t值为-62.789和-12.589,P值均<0.05),72 h后,ATM蛋白水平增高。miR-18a过表达的CNE1和CNE2细胞放疗后与CNE1和CNE2细胞单纯放疗相比,细胞增殖受到明显抑制(P值均<0.05);稳定抑制miR-18a表达与单纯放疗相比,细胞增殖能力增加(P值均<0.01)。100 nmol/L miR-18a模拟物转染联合放疗组与单纯放疗组相比,CNE1细胞凋亡率增加[(22.9±2.1)%比(16.3±1.0)%,t=-4.870,P<0.01]。稳定过表达miR-18a联合放疗与单纯放疗相比,G1期和G2/M期的细胞Objective To explore the impacts of miR-18a overexpression or depression on the radiosensitivities of nasopharyngeal carcinoma cell line CNE1 and CNE2 and underlying mechanisms.Methods CNE1 and CNE2 were transfected with miR-18a mimics,inhibitor and the corresponding control vectors.qRT-PCR and western blot were used to determine the ataxia telangiectasia mutated(ATM)expressions in CNE1 and CNE2.CNE1 and CNE2 with stably expressing miR-18a and miR-18a siRNA were constructed.Methyl thiazolyl tetrazolium(MTT)assay was used to detect the impacts of the miR-18a overexpression or depression combined with irradiation on the cell growth.Flow cytometry was used to detect the cell apoptosis and cell cycle.Colony formation assay was used to evaluate the raodiosensitivities of cells.Acridine orange(AO)staining and western blot were used respectively to test the autophagy and the expressions of related proteins.Independent samples t test was used to compare the mean value between groups by using SPSS 16.0.Results ATM mRNA was decreased significantly in CNE1 and CNE2 cells transfected with 100 or 200 nmol/L miR-18a mimics for 48 hours(CNE1:RQ=0.174±0.139 and 0.003±0.001,t=9.939 and 19470.783;CNE2:RQ=0.024±0.008 and 0.019±0.012,t=270.230 and 137.746,respectively,all P<0.001).ATM proteins were also decreased after transfected with 100 or 200 nmol/L miR-18a mimics for 72 hours.While in the cells transfected with 100 and 200 nmol/L miR-18a inhibitor for 48 hours,the expressions of ATM mRNA were upregulated significantly(CNE1:RQ=9.419±2.495 and 2.500±1.063,t=-4.427 and-41.241;CNE2:RQ=7.210±0.171 and 115.875±15.805,t=-62.789 and-12.589,all P<0.05),and the expressions of ATM proteins increased after transfected for 72 hours.The growth of cells with miR-18a overexpression plus 4 Gy irradiation were obviously inhibited compared to that of cells with the 4Gy irradiation alone;while the growth of miR-18a-inhibited cells increased compared to that of cells with 4 Gy irradiation alone(all P<0.05).CNE1 transfected with 100 nmol/L m

关 键 词：鼻咽肿瘤 miR-18a 放疗敏感性 自噬 

分 类 号：R739.63[医药卫生—肿瘤]