METHODS: Immunocytochemistry, flow cytometry, real-time PCR, western blot, in vitro sphere-formation, and in vivo tumorigenesis assay were used to detect HCC \'stemness\'. Additionally, after forced expression or inhibition of FoxP3, β-catenin expression and HCC \'stemness\' were investigated.
RESULTS: Tregs enhanced the \'stemness\' of HCC cells by upregulating TIC-related markers CD133, Oct3/4, Sox2, c-Myc, Klf4, Nanog, CD13, EpCAM, and inducting epithelial to mesenchymal transition (EMT), increasing TICs ratio, as well as promoting tumorigenic ability. Moreover, β-catenin and c-Myc were upregulated in HCC cells after co-cultured with Tregs. HCC \'stemness\' was inhibited after treatment with Wnt/β-catenin pathway inhibitor. Furthermore, forced expression of FoxP3 resulted in increased GSK3β, decreased β-catenin and TIC ratio in HCC. In contrast, FoxP3 interference reduced GSK3β, enhanced β-catenin and TIC ratio of HCC.
CONCLUSIONS: This study, for the first time, demonstrated that Tregs increased the population of TICs in HCC by inhibiting FoxP3 as well as promoting β-catenin expression.
方法:免疫细胞化学,流式细胞术,实时PCR,westernblot,体外球体形成,和体内肿瘤发生试验用于检测HCC的“干性”。此外,在强制表达或抑制FoxP3后,研究了β-catenin表达和HCC的“干性”。
结果:Tregs通过上调TIC相关标志物CD133,Oct3/4,Sox2,c-Myc,Klf4,Nanog,CD13,EpCAM,并诱导上皮向间充质转化(EMT),增加TIC比率,以及促进致瘤能力。此外,与Tregs共培养后,肝癌细胞中β-catenin和c-Myc表达上调。用Wnt/β-catenin途径抑制剂治疗后,HCC的“干性”受到抑制。此外,FoxP3的强制表达导致GSK3β增加,肝癌中β-catenin和TIC比率降低。相比之下,FoxP3干扰降低了GSK3β,肝癌β-catenin和TIC比值增强。
结论:这项研究,第一次,证明Tregs通过抑制FoxP3和促进β-catenin表达来增加HCC中TIC的数量。