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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 miR-100 on the mitosis and the expression of Polo-like kinase 1 (PLK1) in hepatocellular carcinoma cells. Methods Real-time quantitative PCR and immunofluorescence were used to detect the expression of miR-100 and PLK1 in human hepatoma HepG2 cells. Oligofectamine liposome-mediated Cy3 fluorescent-labeled miR-100 mimics transiently transfected HepG2 cells, cell mitosis and PLK1 protein expression. Results The expression of miR-100 in HepG2 cells was lower than that in normal liver cells, while the expression of PLK1 mRNA and protein was abnormally elevated. At 48h after transfection with miR-100 mimics, the mitotic index of the experimental group was significantly smaller than that of the control cells. Western blotting analysis showed that the PLK1 protein expression in the experimental group was significantly lower than that of the control group. At the same time, immunocytochemistry showed that PLK1 protein located in the nucleus of nucleus in the late stage and the late stage of mitosis basically disappeared. Conclusion miR-100 can inhibit the expression of PLK1 protein and cause mitosis of hepatoma cells.