miR-21、miR-494对黑素瘤A375细胞凋亡和细胞周期的影响

Effect of microRNA-21 and microRNA-494 on cell cycle of and apoptosis in a human melanoma cell line A375

目的探讨miR-21抑制物和miR-494模拟物转染A375细胞的最佳有效转染浓度及其对黑素瘤A375细胞增殖的影响。方法取6个浓度梯度的miR-21抑制物和miR-494模拟物转染A375细胞，实时荧光定量PCR法检测相应的miRNA。继而用Cy5标记的miRNA模拟物阴性对照转染A375细胞，荧光显微镜下观察转染效率。用流式细胞仪检测转染后细胞增殖的变化。结果从A375细胞中成功提出miRNA。定量PCR结果显示，当转染物miR-21抑制物浓度为120nmol／L时，转染细胞内有miR-21模拟物最大程度的下调表达，2^-△△Cr值为0．80（0．65～0．92），低于对照组；当miR-494模拟物浓度为250nmol／L时，转染细胞内有miR-494模拟物最大程度的上调，2^-△△Cr值为126．82（111．52～144．22），明显高于对照组；miR-21抑制物转染组与miR-494模拟物转染组G。期细胞比率分别为（61．61±3．25）％、（61．05±3．17）％，高于对照组（P〈0．05）；增殖指数分别为（38．39±3．25）％、（38．95±3．17）％，低于对照组（P〈0．05）；细胞凋亡率分别为（27．74±1．39）％、（34．30±2．35）％，高于对照组（P〈0．01），细胞转染效率达90％以上。结论miR-21抑制物和miR-494模拟物促使肿瘤细胞的G，期阻滞和促进凋亡作用，miR-21可增强A375细胞增殖，有原癌基因样功能；miR-494可抑制A375细胞增殖，有抑癌基因样作用。

Objective To optimize the concentration of a microRNA-21 （miR-21） inhibitor and a miR-494 mimic for the transfection of A375 human melanoma cells, and to estimate the effect of the miR-21 inbihitor and miR-494 mimic on the proliferation of A375 cells. Methods A miR-21 inbihitor and a miR-494 mimic were designed and constructed. To optimize the concentration of the miR-21 inhihitor and miR-494 mimic for transfection, six concentrations （70 - 250 nmol/L） of the inbihitor and mimic were transfected into A375 cells separately by using Lipofectamine2000. Then, quantitative fluorescence-based PCR was performed to determine the expression of miR-21 and miR-494 in A375 cells. Some A375 cells were classified into five groups： Mock blank control group remaining untransfected, miR-21 inhibitor group transfected with the miR-21 inhibitor, miR-21 control group transfected with the miR-21 inhibitor negative control, miR-494 mimic group transfected with the miR-494 mimic, and miR-494 control group transfected with the miR-494 mimic negative control. After another 48-hour culture, the cells were collected for the analysis of cell apoptosis and cycle by using flow cytometry. Meanwhile, Cy5-1abelled miR-494 mimic negative control was transfected into A375 cells for the evaluation of the transfection efficiency by using an inverted fluorescence microscope. Results miRNAs were successfully extracted from A375 cells. As quantitative PCR revealed, the A375 cells transfected with the miR-21 inhibitor at 120 nmol/L showed the lowest expression level （2^-△△Cr） of miR-21 （average： 0.80; range： 0.65 - 0.92）, and those transfected with the miR- 494 mimic at 250 nmal/L displayed the highest expression level of miR-494 （average： 126.82; range： 111.52 - 144.22）. The transfection efficiency in A375 cells was higher than 90%. Compared with the corresponding negative control groups, the miR-21 inhibitor group and miR-494 mimic group showed increased apoptosis rate （（27.74± 1.39）% vs. （12.93 ± 0.65）%, （34.30 ±2.35）% vs. （15.54 ± 1.02）%, both P 〈 0.01）, percentage of Gl-phase cells （（61.61 ±3.25）% vs. （50.34 ± 5.62）%, （61.05 ± 3.17）% vs. （49.95 ±2.58）%, both P 〈 0.05）, but decreased proliferation index （（38.39± 3.25）% vs. （49.66 ± 5.62） %, （38.95 ± 3.17）% vs. （50.05± 2.58）%, both P 〈 0.05 ）. Conclusions Both the miR-21 inhibitor and miR-494 mimic can promote the Gl-phase arrest and apoptosis in A375 cells, and miR-21 may act as a protooneogene accelerating the proliferation of A375 cells, while miR-494 may founetion as a tumor suppressor inhibiting the proliferation of A375 cells.