慢病毒介导的microRNA-155过表达对肝癌细胞HepG2增殖的影响

摘要： 目的 通过构建慢病毒 （LV） 介导的miRNA-155 （miR-155） 过表达载体， 探讨上调miR-155对肝癌细胞系 HepG2增殖的影响。方法 针对目标序列合成特异性miR-155过表达片段并克隆入慢病毒载体pGCSHL-GFP， 将重组miR-155-hRNA-LV和阴性对照空病毒载体转染HepG2细胞， 建立稳定过表达miR-155的细胞系。细胞设过表达组 （H组）、 阴性对照组 （negative control， NC组） 和正常组 （normal， N组）。实时荧光聚合酶链式反应 （qRT-PCR） 方法检测各组miR-155和PTEN的表达水平； Western blot检测各组PTEN、 CyclinD1、 CyclinA1+A2蛋白表达情况； 细胞增殖-毒性检测 （CCK-8） 细胞增殖情况。结果 H组miR-155表达量明显高于其NC组及N组， 靶基因PTEN的表达量低于NC组及N组 （均P＜0.05）。H组PTEN蛋白的表达量明显低于NC组和N组， 而CyclinD1、 CyclingA1+A2蛋白的表达量明显高于NC组和N组 （均P＜0.05）。CCK-8结果显示3组12 h时吸光度值开始出现差异， 24 h、 48 h、 72 h H 组吸光度值均明显大于NC组和N组 （P＜0.05）， 但后2组差异无统计学意义。结论 过表达miR-155可促进肝癌细胞系HepG2细胞增殖。

The effect of lentivirus-mediated overexpression of microRNA-155 on hepatoma cell HepG2 proliferation

Abstract：Objective To investigate the effect of up-regulation of microRNA-155 (miR-155) on the proliferation of hepatoma cell line HepG2 by constructing lentivirus (LV) overexpressing miRNA-155. Methods The specific miR-155 overexpressing fragment was synthesized into the target sequence and cloned into the lentiviral vector pGCSHL-GFP. The recombinant miR-155-hRNA-LV and the negative control empty viral vector were transfected into HepG2 cells to establish stable overexpression miR-155 cell line. The miR-155 cells were divided into three groups including the overexpression group (H group), the negative control group (negative control, NC group) and the normal group (normal, N group). Real-time fluorescent polymerase chain reaction (qRT-PCR) was used to detect the expression levels of miR-155 and PTEN in three groups. Western blot assay was used to analyze relative expressions of PTEN, CyclinD1 and CyclinA1+A2 proteins in three groups. Cell proliferation-toxicity test (CCK-8) was used to detect cell proliferation. Results The expression of miR-155 was significantly higher in H group than that in NC group and N group. The expression of target gene PTEN was lower in H group than that in NC group and N group (both P＜0.05). The expression of PTEN protein was significantly lower in H group than that in NC group and N group, while the expressions of CyclinD1 and CyclingA1+A2 protein were significantly higher in H group compared with that of NC group and N group (both P＜0.05). CCK-8 results showed that the photometric values of the three groups began to differ at 12 h, and the luminosity values were significantly greater in H group than those of NC group and N group from 24 h to 72 h, but the difference was not statistically significant between NC group and N group. Conclusion Overexpression of miR-155 can promote the proliferation of HepG2 cells.