TGF-β诱导肝癌细胞向肿瘤干细胞分化的研究

摘要

目的 探讨转化生长因子-β(TGF-β)诱导肝癌细胞向肿瘤干细胞分化的机制.方法 TGF-β诱导肝癌细胞(HepG2细胞)上皮间质化(EMT),荧光定量PCR检测肝癌细胞EMT指标变化.流式细胞术、蛋白免疫印迹法及荧光定量PCR检测肝癌细胞受诱导后干细胞标志物EPCAM变化,并检测Wnt通路的β-catenin变化及激光共聚焦检测β-catenin入核情况.蛋白免疫印迹法检测β-catenin沉默后干细胞标志物上皮细胞黏附分子(EPCAM)的变化.结果 TGF-β可诱导肝癌细胞形态的改变、上皮细胞标志物E-cadherin下调和间质细胞标志物Vimentin上调,肝癌干细胞标志物EPCAM上调.TGF-β可能通过激活Wnt通路β-catenin的蛋白水平和mRNA水平,以及促进其核转录诱导肝癌细胞向干细胞分化.沉默β-catenin可以抑制TGF-β诱导肝癌细胞向干细胞分化的过程.结论 TGF-β可通过激活Wnt/β-catenin通路诱导肝癌细胞向肿瘤干细胞分化.%Objective To investigate the mechanism underlying the effect of transforming growth factor-β(TGF-β) on inducing hepatocellular carcinoma cells differentiating to cancer stem cells.Methods TGF-β induced the epithelial-to-mesenchymal transition (EMT) of the hepatocellular carcinoma cells (HepG2 cells).The changes of the relevant parameters during the EMT of HepG2 cells were detected by quantitative fluorescent polymerase chain reaction (PCR).Flow cytometry,western blot and quantitative fluorescent PCR were performed to investigate the changes of the expression of epithelial cellular adhesion molecule (EPCAM) and β-catenin in the Wnt signaling pathway after TGF-β induction.Confocal microscopy analysis was used to detect the nuclear translocation of β-catenin.The changes of the expression of EPCAM after β-catenin silencing were investigated by western blot.Results TGF-βinduction could cause the morphological changes of HepG2 cells,down-regulate the expression of epithelial cell marker E-cadherin,up-regulate the expression of mesenchymal cell marker Vimentin and up-regulate the expression of cancer stem cell marker EPCAM.TGF-β could induce the hepatocellular carcinoma cells differentiating into cancer stem cells probably by up-regulating the expression levels of β-catenin protein and mRNA in the Wnt signaling pathway.Silencing of β-catenin could suppress the TGF-β inducing differentiation of hepatocellular carcinoma cells into cancer stem cells.Conclusions TGF-β can induce the differentiation of hepatoellular carcinoma cells into cancer stem cells by activating the Wnt/β-catenin signaling pathway.