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

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（80 μmol/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；Annexin Ⅴ-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；80 μmol/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，80 μmol/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，80 μmol/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；Annexin Ⅴ-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。

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

Objective To investigate the effect of down-regulation of miR-205-5p on 3-bromopyruvate-induced apoptosis inhuman nasopharyngeal carcinoma CNE2Z cells. Methods Nasopharyngeal carcinoma CNE2Z cells were transfected withmiR- 205-5p-mimic or miR-205-5p-inhibitor, treated with 80 μmol/L 3-bromopyruvate alone, or exposed to both of thetreatments. The proliferation of the treated cells was examined with MTT assay, and early apoptosis of the cells was detectedusing a mitochondrial membrane potential detection kit (JC-1). DAPI fluorescence staining was used to detect morphologicalchanges of the cell nuclei and late cell apoptosis; Annexin V-FITC/PI double staining was employed to detect the cell apoptosisrate. Western blotting was used to detect the expressions of Bcl-2, Bax, Mcl-1 and Bak proteins. Results Exposure to3-bromopyruvate significantly inhibited the proliferation of CNE2Z cells, and increasing the drug concentration and extendingthe treatment time produced stronger inhibitory effects. Treatment with 80 μmol/L 3-bromopyruvate for 24, 48 and 72 hresulted in inhibition rates of (45.7±1.21)%, (64.4±2.02)% and (78.3±1.55)% in non-transfected CNE2Z cells, respectively; theinhibition 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 ofmitochondrial membrane potential detection showed that the relative proportion of red and green fluorescence decreasedsignificantly in miR-205-5p-inhibitor-transfected cells with 3-bromopyruvate treatment. Combined treatment of the cellswith 3-bromopyruvate and miR-205-5p-inhibitor transfection obviously increased nuclear fragmentation and nuclear pyknosisand significantly increased cell apoptotic rate ascompared with the two treatments alone (P<0.01),causing also decreased expressions of Bcl-2 and Mcl-1proteins and increased expressions of Bax and Bakproteins. Conclusion Inhibition of miR-205-5p enhancesthe proapototic effect of 3-bromopyruvate in CNE2Z cellspossibly in relation to the down-regulation of Mcl-1 andBcl-2 and the up-regulation of Bak and Bax proteins.