微小RNA-27 a对黑色素瘤WM239细胞增殖和凋亡的影响

Effect of miR-27a on cell proliferation and apoptosis in human melanoma cell line WM239

目的：探讨微小RNA-27a（ miR-27a）模拟物和抑制物转染黑色素瘤WM239细胞后对细胞增殖和凋亡的影响。方法将miR-27a模拟物、抑制物及其阴性对照转染WM239细胞，荧光显微镜观察转染效率，实时荧光定量PCR检测相应的微小RNA，四甲基偶氮唑盐（ MTT）法检测细胞增殖，流式细胞仪检测细胞凋亡和细胞周期。结果细胞转染效率为80%~90%。转染miR-27a模拟物后，细胞内miR-27a表达量明显上升（2-△△CT值为26.98±0.01），与正常对照组相比差异有统计学意义（ t＝－1123.67，P＝0.00）；转染miR-27a抑制物后，细胞中miR-27a的表达量下降（2-△△CT值为0.96±0.02），与正常对照组相比差异无统计学意义（t＝4.04，P＝0.06）。转染miR-27a模拟物后，细胞增殖受到明显抑制，与正常对照组相比差异具有统计学意义[72 h吸光度（0.45±0.02）∶（0.72±0.01），F＝129.56，P﹤0.05]。miR-27a模拟物组G0-G1期的细胞比例升高[（74.83±1.46）∶（63.73±1.25），F＝30.33，P﹤0.05]，S期和G2-M期细胞比例减少[（21.33±1.75）∶（27.50±1.25），F＝14.98，P﹤0.05；（3.90±1.31）∶（8.80±2.10），F＝3.66，P﹤0.05]；模拟物组细胞凋亡率与正常对照组相比明显增加[（29.67±0.91）%∶（1.44±0.85）%， F＝530.90，P﹤0.01]；而抑制物组对细胞周期和凋亡无明显作用。结论 miR-27a抑制黑色素瘤细胞增殖，具有抑瘤作用，这与其促进细胞凋亡，阻滞细胞周期于G0-G1期相关。

Objective To investigate the effect of miR-27a mimic and inhibitor on proliferation and apoptosis in melanoma cell line WM239. Methods The miR-27a mimic,inhibitor and its negative control were transfected into WM239 cells. The transfection efficiency was evaluated by fluorescence microscope. The expres-sion of miR-27a was detected by real-time fluorescent quantitative PCR. The proliferation of cells was detected by MTT. The cell apoptosis and cell cycle were analyzed by flow cytometry. Results The transfection efficiency in WM239 cells was 80% to 90%. The expression of miR-27a was markedly up-regulated in miR-27a mimic group （2-△△CT value is 26. 98 ± 0. 01）,with statistically significant difference（t= -1 123. 67,P=0. 00）;and the miR-27a inhibitor group showed lower expression of miR-27a（2-△△CT value is 0. 96 ± 0. 02）,there was no statisti-cally significant difference compared with normal control group（t=0. 04,P=0. 06）. The proliferation of cells was obviously inhibited in miR-27a mimic group,and there was statistically significant difference compared with normal control group[absorbance of 72 h（0. 45 ± 0. 02）∶（0. 72 ± 0. 01）,F=129. 56,P﹤0. 05]. The percent-age of WM239 cells in G0-G1 phase was increased[（74. 83 ± 1. 46）∶（63. 73 ± 1. 25）,F=30. 33,P﹤0. 05], and the percentage of WM239 cells in S phase and G2-M phase were decreased[（21. 33 ± 1. 75）∶（27. 50 ± 1. 25）,F=14. 98,P﹤0. 05;（3. 90 ± 1. 31）∶（8. 80 ± 2. 10）,F=3. 66,P﹤0. 05]. The apoptosis rate of cells＆amp;nbsp;was significantly increased in miR-27a mimic group compared with normal group[（29. 67 ± 0. 91）%∶（1. 44 ± 0. 85）%,F=530. 90,P﹤0. 01],but the inhibitor group had no obvious effect on cell cycle and cell apoptosis. Conclusion MiR-27a can suppress melanoma cell proliferation and act as a tumor suppressor gene,which is rel-evant to induce cell apoptosis and block cell cycle in G0-G1 phase.