微小RNA-221对HepG2细胞增殖及其凋亡的影响

目的 探讨微小RNA-221(miR-221)对肝癌细胞株HepG2细胞增殖与凋亡的影响.方法 将miR-221阻遏物及模拟物转染至HepG2后,用实时荧光定量RT-PCR检测miR-221的表达水平;用细胞增殖试剂盒、Hoechst 33342及碘化丙啶(PI)双重染色、流式细胞仪及caspase3/7活性试剂盒检测HepG2细胞增殖与凋亡情况.对数据进行多样本的单因素方差分析,两两比较采用LSD法;相关性比较用Pearson检验.结果 RT-PCR结果显示,转染miR-221阻遏物后其表达受到抑制,而转染miR-221模拟物可促进其表达.细胞增殖试剂盒及Hoechst 33342/PI染色结果显示,48 h后miR-221阻遏物明显抑制HepG2细胞增殖(P＜0.05),而miR-221模拟物促进HepG2细胞增殖(P＜0.05),两种方法结果呈正相关(r=0.993,P＜0.01).流式细胞仪检测细胞周期显示,miR-221模拟物组G1期细胞比例(47.6%±1.53%)明显低于空白对照组(59.00%±1.00%)及阴性对照组(58.00%±1.00%,F=81.77,P＜0.01); S期细胞比例(20.33%±1.15%)明显高于空白对照组(11.00%±1.00%)及阴性对照组(12.00%±1.00%,F=70.90,P＜0.01).Hoechst 33342/PI染色、流式细胞仪膜联蛋白V凋亡试剂盒检测均显示,转染miR-221阻遏物48 h后,细胞凋亡和坏死增加(P＜0.05),差异有统计学意义.Caspase-3/7试剂盒检测caspase-3/7活性变化结果显示,转染miR-221阻遏物24 h后,细胞caspase3/7活性明显增高(P＜0.05),差异有统计学意义.结论 miR-221可促进肝癌细胞生长增殖,抑制miR-221表达可诱导细胞凋亡.miR-221有望成为治疗肝癌新的分子靶点之一.

Effect of miR-221 on the viability and apoptosis of hepatocellular carcinoma HepG2 cells

Objective To investigate the effect of microRNA-221 (miR-221) on cell viability and apoptosis ofhepatocellular carcinoma HepG2 cells. Methods MiR-221 inhibitors and mimics were transfected into HepG2 cells. The expression of miR-221 was detected by real time quantitative RT-PCR. CellTiter-blue cell viability kit, Hoechst 33342/propidium iodide (PI) double staining assay, flow cytometry and Apo-ONE homogeneous caspase-3/7 kit were used to measure cell viability and apoptosis. Results MiR-221 inhibitors significantly inhibited the cell growth and miR-221 mimics increased cell viability 48 hours post-transfection measured by both CellTiter-blue cell viability kit and Hoechst 33342/PI assay (P ＜ 0.05). There was a positive correlation between these two assays(r = 0.993, P ＜ 0.01). With miR-221 mimics, the number of G1 stage cells (47.67% ± 1.53%) was significantly reduced as compared to the blank control (59.00% ± 1.00%) and the negative control (58.00% ± 1.00%, F= 81.77, P ＜ 0.01), and it was significantly raised in S stage (20.33% ± 1.15%) than in blank control ( 11.00% ± 1.00%) and negative control (12.00% ± 1.00%, F = 70.9, P ＜ 0.01) with flow cytometry analysis. More cell apoptosis and necrosis were significantly induced by miR-221 inhibitors 48 hours post-transfection detected by both Hoechst 33342/PI assay and flow cytometry PE Annexin V kit (P ＜ 0.05).The result from Apo-ONE homogeneous caspase-3/7 kit was consistent with the above two apoptotic assays,which showed that with miR-221 inhibitors, the activity of caspase-3/7 was significantly enhanced (P ＜ 0.05).Conclusions MiR-221 contributes to the growth of hepatocellular carcinoma cells and miR-221 inhibition can induce cell apoptosis. miR-221 has the potential to become one of the new molecular targets for liver cancer therapy.