下调miR-205-5p增强3-溴丙酮酸对人鼻咽癌CNE2Z细胞的凋亡诱导作用  

Down-regulation of miR-205-5p enhances pro-apoptotic effect of 3-bromopyruvate on human nasopharyngeal carcinoma CNE2Z cells

作　　者：时宗芬 张配 鲁星月 朱晨露 陈长江 赵素容 刘浩 SHI Zongfen;ZHANG Pei;LU Xingyue;ZHU Chenlu;CHEN Changjiang;ZHAO Surong;LIU Hao(School of Pharmacy,Bengbu Medical College/Anhui Provincial Engineering Technology Research Center of Biochemical Pharmaceuticals,Bengbu 233030,China)

机构地区：[1]蚌埠医学院药学院//安徽省生化药物工程技术研究中心

出　　处：《南方医科大学学报》2019年第10期1166-1172,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(81603155);安徽省自然科学基金青年基金(1708085QH212);安徽省高等学校自然科学研究重点项目(KJ2016A486);安徽省大学生创新创业训练项目(201710367061)~~

摘　　要：目的探讨下调miR-205-5p对3-溴丙酮酸诱导的鼻咽癌CNE2Z细胞凋亡的影响。方法以鼻咽癌CNE2Z细胞株为研究对象,实验分为4组,分别为对照组、转染组(转染miR-205-5p-mimic或miR-205-5p-inhibitor)、3-溴丙酮酸组(80μmol/L 3-溴丙酮酸)、合用组(转染miR-205-5p-mimic或miR-205-5p-inhibitor后合用3-溴丙酮酸)。MTT法检测3-溴丙酮酸和miR-205-5p对CNE2Z细胞增殖的影响;线粒体膜电位检测试剂盒(JC-1)检测细胞早期凋亡情况;DAPI荧光染色法检测细胞核形态变化以及细胞晚期凋亡情况;AnnexinⅤ-FITC/PI双染法检测细胞凋亡率的变化;Western blot检测Bcl-2、Bax、Mcl-1、Bak蛋白的表达。结果3-溴丙酮酸对CNE2Z细胞的增殖有明显的抑制作用,并且随着药物浓度和作用时间的增加细胞增殖抑制作用越明显;80μmol/L 3-溴丙酮酸处理CNE2Z细胞24、48、72 h的抑制率分别为(45.7±1.21)%、(64.4±2.02)%、(78.3±1.55)%;转染miR-205-5p-mimic后,80μmol/L 3-溴丙酮酸作用24、48、72 h的抑制率分别为(27.7±1.04)%、(34.8±2.10)%、(44.3±1.57)%;转染miR-205-5p-inhibitor后,80μmol/L 3-溴丙酮酸作用24、48、72 h的抑制率分别为(80.5±0.94)%、(87.9±0.50)%、(93.8±1.16)%。线粒体膜电位检测结果显示,转染miR-205-5p-inhibitor后3-溴丙酮酸组红绿荧光的相对比例明显降低;DAPI荧光检测结果显示,转染miR-205-5p-inhibitor后3-溴丙酮酸组的细胞核碎裂及核固缩明显增加;AnnexinⅤ-FITC/PI双染法检测结果显示,转染miR-205-5p-inhibitor后3-溴丙酮酸组的凋亡率明显高于单独处理组的凋亡率(P<0.01);Western blot检测结果显示,转染miR-205-5p-inhibitor后3-溴丙酮酸组的Bcl-2、Mcl-1蛋白的表达水平明显降低,Bax、Bak蛋白的表达水平明显增高。结论3-溴丙酮酸能诱导CNE2Z细胞凋亡,转染miR-205-5p-inhibitor能增强3-溴丙酮酸诱导的细胞凋亡,其机制可能与下调Mcl-1和Bcl-2表达,上调Bak和Bax蛋白的表达有关。Objective To investigate the effect of down-regulation of miR-205-5p on 3-bromopyruvate-induced apoptosis in human nasopharyngeal carcinoma CNE2Z cells.Methods Nasopharyngeal carcinoma CNE2Z cells were transfected with miR-205-5p-mimic or miR-205-5p-inhibitor,treated with 80μmol/L 3-bromopyruvate alone,or exposed to both of the treatments.The proliferation of the treated cells was examined with MTT assay,and early apoptosis of the cells was detected using a mitochondrial membrane potential detection kit(JC-1).DAPI fluorescence staining was used to detect morphological changes of the cell nuclei and late cell apoptosis;Annexin V-FITC/PI double staining was employed to detect the cell apoptosis rate.Western blotting was used to detect the expressions of Bcl-2,Bax,Mcl-1 and Bak proteins.Results Exposure to 3-bromopyruvate significantly inhibited the proliferation of CNE2Z cells,and increasing the drug concentration and extending the treatment time produced stronger inhibitory effects.Treatment with 80μmol/L 3-bromopyruvate for 24,48 and 72 h resulted in inhibition rates of(45.7±1.21)%,(64.4±2.02)%and(78.3±1.55)%in non-transfected CNE2Z cells,respectively;the inhibition rates were(27.7±1.04)%,(34.8±2.10)%and(44.3±1.57)%in the cells transfected with miR-205-5p-mimic,and were(80.5±0.94)%,(87.9±0.50)%and(93.8±1.16)%in cells transfected with miR-205-5p-inhibitor,respectively.The results of mitochondrial membrane potential detection showed that the relative proportion of red and green fluorescence decreased significantly in miR-205-5p-inhibitor-transfected cells with 3-bromopyruvate treatment.Combined treatment of the cells with 3-bromopyruvate and miR-205-5p-inhibitor transfection obviously increased nuclear fragmentation and nuclear pyknosis and significantly increased cell apoptotic rate as compared with the two treatments alone(P<0.01),causing also decreased expressions of Bcl-2 and Mcl-1 proteins and increased expressions of Bax and Bak proteins.Conclusion Inhibition of miR-205-5p enhances the proapototic

关 键 词：鼻咽癌 miR-205-5p 3-溴丙酮酸 细胞凋亡 

分 类 号：R73[医药卫生—肿瘤]