摘要
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，48h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（47．67％±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48h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24h后，细胞caspase3／7涪陡明显增高（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。
Objective To investigate the effect ofmicroRNA-221 （miR-221） on cell viability and apoptosis of hepatocellular 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 48hours 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 molecttlar targets for fiver cancer therapy.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2011年第8期582-587,共6页
Chinese Journal of Hepatology
基金
广西自然科学基金（桂科青1013059）
