MiR-100对肝癌细胞有丝分裂阻滞及Polo样激酶1蛋白表达的作用

Effects of microRNA-100 on mitosis block and Polo-like kinase 1 expression in hepatocellular carcinoma cells

目的探讨microRNA-100(miR-100)对肝癌细胞有丝分裂阻滞及Polo样激酶1(PLK1)表达的作用。方法采用实时定量PCR和免疫荧光方法检测miR-100和PLK1在人肝癌细胞HepG2中的表达。利用Oligofectamine脂质体介导将Cy3荧光标记的miR-100模拟物瞬时转染HepG2细胞,分析细胞有丝分裂和PLK1蛋白表达情况。结果肝癌细胞HepG2的miR-100表达量低于正常肝细胞,而PLK1 mRNA和蛋白表达却异常升高。在miR-100模拟物转染后48h,实验组细胞有丝分裂指数显著小于对照细胞,经Western blotting分析显示,实验组细胞PLK1蛋白表达水平较对照组细胞均显著减低。同时免疫细胞化学检测发现,在有丝分裂中晚期和末期位于细胞核内的PLK1蛋白基本消失。结论 miR-100具有抑制PLK1蛋白表达的作用,可引起肝癌细胞有丝分裂阻滞。

Objective To investigate the effect of microRNA-100 （miR-100） on mitosis block and Polo-like kinase 1 （PLK1） expression in hepatocellular carcinoma cells. Methods Expression of miR-100 and PLK1 was determined by quantitative real-time PCR （qRT-PCR） and immunofluorescence （IF） in hepatocellular carcinoma HepG2 cells. HepG2 cells were transiently transfected by miR-100 mimic with Cy3 fluorescence labeled through Oligofectamine liposomes, followed by cell mitosis and PLK1 expression analysis. Results Expression level of miR-100 decreased whereas PLK1 mRNA and protein expression abnormally increased in HepG2 cells compared with the normal hepatocytes. At 48hours after transfection of miR-100 mimic, the mitotic index of HepG2 cells in experimental group was significantly less than that of control cells. Western blotting analysis indicated that PLK1 protein expression levels were significantly reduced in experimental cells compared with the control ceils. Immunocytochemical results showed that PLK1 protein in the nucleus almost disappeared in meta/ana-phase and telophase of mitosis. Conclusion MiR-100 may inhibit the expression of PLK1 protein, which leads to mitotic block of hepatocellular carcinoma cells.