Transforming growth factor-β (TGF-β) is a multifunctional regulator of cell growth, apoptosis, differentiation and migration. The Wnt/β-catenin signaling pathway has been implicated in a wide spectrum of diseases, including numerous cancers and degenerative disease. The aim of the present study was to investigate if simultaneous blocking of TGF-β and Wnt/β-catenin signaling pathways exerts synergistic anti-tumor effects on hepatocellular carcinoma (HCC) cells. Short hairpin (sh) RNA eukaryotic expression vectors, specific to TGF-β receptor II (RII) and Frizzled receptor (FZD)-7, were constructed by gene recombination. The expression vectors were transfected into human HCC HepG2 and Huh-7 cells using Lipofectamine 2000 to investigate the synergistic effects between TGF-β and Wnt/β-catenin signaling pathways on HCC cell proliferation, invasion and migration and the cell-cycle distribution. Western blot analysis was used to identify the expression of β-catenin, c-Myc and cyclin D1 in transfected cells to investigate the underlying mechanisms that cause TGF-β and Wnt/β-catenin signaling in HCC cells. shTGF-βRII-c and shFZD-7-2 were selected as the most efficient plasmids. A cell growth assay and colony-forming assay consistently demonstrated that the proliferative activity of the co-transfected group was significantly decreased compared to the single-transfected group. A wound healing invasion and migration assay demonstrated that co-transfection of shTGF-βRII-c and shFZD-7-2 decreased the invasion and migration abilities of the cells compared with either single-transfected group. In addition, the present study demonstrated that the observed reduction in cell proliferation was due to the cells arresting at the G1 phase of the cell cycle, and the downregulation of β-catenin, c-Myc and cyclin D1 impaired the proliferative and invasive abilities of the HCC cells. The present results demonstrate that simultaneous blocking of TGF-β and Wnt/β-catenin signaling by targeting TGF-βRII and FZD-7 may inhibit the proliferation and metastasis of HCC cells more effectively compared with blocking either the TGF-β or Wnt/β-catenin pathway.
Keywords: TGF-β signaling; Wnt/β-catenin signaling; human hepatocellular carcinoma; metastasis; proliferation; synergistic effects.