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.
Nan Fang Yi Ke Da Xue Xue Bao. 2019 Oct 30;39(10):1166-1172. doi: 10.12122/j.issn.1673-4254.2019.10.06.
To investigate the effect of down-regulation of miR-205-5p on 3-bromopyruvate-induced apoptosis in human nasopharyngeal carcinoma CNE2Z cells.
Nasopharyngeal carcinoma CNE2Z cells were transfected with miR- 205-5p-mimic or miR-205-5p-inhibitor, treated with 80 μmol/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.
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 80 μmol/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 ( < 0.01), causing also decreased expressions of Bcl-2 and Mcl-1 proteins and increased expressions of Bax and Bak proteins.
Inhibition of miR-205-5p enhances the proapototic effect of 3-bromopyruvate in CNE2Z cells possibly in relation to the down-regulation of Mcl-1 and Bcl-2 and the up-regulation of Bak and Bax proteins.
探讨下调miR-205-5p对3-溴丙酮酸诱导人鼻咽癌CNE2Z细胞凋亡的影响。
将miR-205-5p模拟物或miR-205-5p抑制剂转染至鼻咽癌CNE2Z细胞，单独用80μmol/L 3-溴丙酮酸处理，或同时进行这两种处理。用MTT法检测处理后细胞的增殖情况，使用线粒体膜电位检测试剂盒（JC-1）检测细胞早期凋亡。用DAPI荧光染色检测细胞核形态变化和细胞晚期凋亡；采用Annexin V-FITC/PI双染法检测细胞凋亡率。用蛋白质印迹法检测Bcl-2、Bax、Mcl-1和Bak蛋白的表达。
3-溴丙酮酸处理显著抑制CNE2Z细胞的增殖，增加药物浓度和延长处理时间产生更强的抑制作用。用80μmol/L 3-溴丙酮酸处理24、48和72 h，未转染的CNE2Z细胞的抑制率分别为（45.7±1.21）%、（64.4±2.02）%和（78.3±1.55）%；转染miR-205-5p模拟物的细胞的抑制率分别为（27.7±1.04）%、（34.8±2.10）%和（44.3±1.57）%，转染miR-205-5p抑制剂的细胞的抑制率分别为（80.5±0.94）%、（87.9±0.50）%和（93.8±1.16）%。线粒体膜电位检测结果显示，3-溴丙酮酸处理的转染miR-205-5p抑制剂的细胞中，红色和绿色荧光的相对比例显著降低。与单独的两种处理相比，3-溴丙酮酸与miR-205-5p抑制剂转染联合处理细胞明显增加了核碎裂和核固缩，显著提高了细胞凋亡率（P<0.01），还导致Bcl-2和Mcl-1蛋白表达降低，Bax和Bak蛋白表达增加。
抑制miR-205-5p可增强3-溴丙酮酸对CNE2Z细胞的促凋亡作用，可能与下调Mcl-1和Bcl-2以及上调Bak和Bax蛋白有关。
