β-catenin基因沉默对肝癌干细胞干性转录因子的影响

Effects of beta-catenin gene silencing on stemness transcription factors in hepatocellular carcinoma stem cells

背景:肝癌干细胞是导致癌细胞形成、更新和增殖的原因,寻找针对肝癌干细胞治疗的关键靶点,并进一步拓展肝癌综合治疗手段是重要研究课题。目的:观察β-catenin基因沉默后CD133+HepG2细胞干性转录因子变化,探讨β-catenin调控肝癌干细胞干性的作用机制。方法:采用免疫磁珠筛选CD133+HepG2细胞,然后经干细胞培养基诱导、扩增方案获得CD133+HepG2肝癌干细胞,流式细胞术鉴定CD133+HepG2细胞阳性率,RNA干扰技术构建沉默β-catenin基因表达的β-catenin-shRNA慢病毒质粒,转染CD133+HepG2肝癌干细胞72 h,荧光标记并评估转染效率。确认β-catenin沉默后,平板克隆形成实验观察CD133+HepG2肝癌干细胞增殖情况,流式细胞术检测CD133+HepG2肝癌干细胞的细胞周期,qRT-PCR和Western blot检测干性转录因子SOX2,NANOG,OCT4的mRNA及蛋白表达水平。结果与结论:与空白对照组和病毒空载组相比,病毒干扰组的细胞克隆形成能力明显降低,G_(0)/G_(1)期细胞百分比明显增多,S期和G_(2)/M期细胞百分比降低,NANOG、SOX2、OCT4的蛋白和mRNA表达水平明显降低(P<0.05),提示下调β-catenin表达可能对抑制肝癌干细胞的干性水平具有潜在作用。

BACKGROUND:Hepatocellular carcinoma stem cell is responsible for the formation,renewal and proliferation of cancer cells.It is a major topic to find key targets for hepatocellular carcinoma stem cell therapy and to further expand comprehensive treatment means for liver cancer.OBJECTIVE:To observe changes of stemness transcription factors afterβ-catenin gene silencing on CD133+HepG2 cells,and investigate the action mechanism ofβ-catenin regulating stemness of hepatocellular carcinoma stem cells.METHODS:After CD133+HepG2 hepatocellular carcinoma stem cells were screened by magnetic activated cell sorting,these cells were induced and cultured in stem cell culture medium.Positive rate of CD133+HepG2 hepatocellular carcinoma stem cells was identified by flow cytometry.β-Catenin shRNA lentiviral plasmid was constructed to inhibitβ-catenin expression by RNA interference.After transfected CD133+HepG2 hepatocellular carcinoma stem cells for 72 hours,plasmid was labeled with fluorescence,which evaluated transfection efficiency.Afterβ-catenin silencing was confirmed,CD133+HepG2 hepatocellular carcinoma stem cell proliferation was measured by plate clone formation assay.CD133+HepG2 hepatocellular carcinoma stem cell cycle was detected by flow cytometry.RT-PCR and western blot assay were used to detect the mRNA and protein expression of stemness transcription factors SOX2,NANOG and OCT4.RESULTS AND CONCLUSION:Compared with blank control group and virus-free group,clone formation ability was decreased in virus-blockade group.Percentage of G_(0)/G_(1) phase cells increased significantly,while that of S phase cells and G_(2)/M phase cells decreased.Protein and mRNA expression of NANOG,SOX2 and OCT4 was significantly decreased in virus-blockade group(P<0.05).It is indicated that down-regulation ofβ-catenin expression may have a potential effect on suppressing the stemness of hepatocellular carcinoma stem cells.