肝癌细胞微小RNA-100对细胞有丝分裂的影响

Eect of microRNA-100 on mitosis in hepatocellular carcinoma cells

目的:探讨微小RNA(micro RNA-100,miR-100)对人肝癌HepG2细胞有丝分裂的影响。方法:采用实时荧光定量PCR法检测HepG2细胞中miR-100和Polo样激酶1(Polo-like kinase1,Plk1)mRNA的相对表达水平。以脂质体介导的方法将化学合成的带有Cy3荧光标记的miR-100模拟物瞬时转染至HepG2细胞,采用FCM法检测磷酸化组蛋白H3(phospho histone H3,PHH3)标志的转染后细胞的有丝分裂指数,光学显微镜观察细胞的有丝分裂情况,免疫细胞化学法分析转染后细胞中Plk1蛋白表达的变化。结果:同正常肝细胞比较,肝癌HepG2细胞的miR-100水平降低(P<0.05),而Plk1mRNA水平明显升高(P<0.01)。在转染miR-100模拟物48h后,显微镜下可见有丝分裂细胞数较对照组明显减少,且细胞有丝分裂指数明显低于对照组细胞[(0.62±0.48)%vs(10.34±0.77)%,P<0.01]。同对照组相比,miR-100转染组细胞中Plk1mRNA水平在转染后48h时显著降低(P<0.01),且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:肝癌细胞中miR-100水平升高可导致细胞有丝分裂阻滞,这可能与细胞核内Plk1蛋白缺失密切相关。

Objective： To investigate the effect of miR-100 （microRNA-100） on mitosis of human hepatoma HepG2 cells. Methods： The relative levels of miR-100 and Plk1 （Polo-like kinase 1） mRNA in HepG2 cells were detected by RFQ-PCR （real-time fluorescence quantitative PCR）. Then the HepG2 cells were transiently transfected with liposomes retaining miR-100 mimic which was chemically synthesized and labeled with Cy3 fluorescent marker. After transfection, the activity of mitosis was observed by microscopy and detected by FCM （flow cytometry） using PPH3 （phosphohistone H3） antibody. Moreover, the expression of Plk1 protein was examined by ICC （immunocytochemistry）. Results： As compared with the normal hepatocytes, the miR-100 level was decreased in HepG2 cells （P 〈 0.05）, but the expression of Plk1 mRNA was increased （P 〈 0.01）. At 48 h after transfection of miR-100 mimic, the number of mitotic cells in the experimental group was significantly reduced comparing with that of in the control group （P 〈 0.01）, and the mitotic index of the experimental group was significantly reduced comparing with that of the untransfected control group [（0.62 ± 0.48）% vs （10.34 ± 0.77）%, P 〈 0.01]. The expression level of Plk1 mRNA in HepG2 cells was significantly reduced at 48 h after transfection with miR-100 comparing with those of the other groups （P 〈 0.01）,and the Plk1 protein locating in the nucleus almost disappeared in metaphase, anaphase, and telophase of mitosis. Conclusion： The increased level of miR-100 can lead to mitotic block in hepatoma cells, which may be closely associated with the loss of Plk1 protein expression in nucleus.