Figure 4

Upregulation of DKK1 promotes the expression of β-catenin. A: The representative images of IHC staining for β-catenin. Scale bar = 50 μm. B: β-catenin expression is positively correlated with DKK1 level in 71 human HCC tissues. The median of all 71 cases was chosen as the cutoff point for separating low-DKK1 (n = 36) from high-DKK1 expressing tumors (n =35). C: DKK1 overexpression markedly enhanced the mRNA and protein level of β-catenin in HepG2 cells. GAPDH and β-actin served as internal controls. D: up-regulation of DKK1 expression enhanced the cytoplasmic/nuclear β-catenin accumulation in HepG2 tumor cells (original magnification, 100×). The expression level of β-catenin protein was detected with rabbit polyclonal antibody and revealed by Cy3-conjugated secondary antibody (red). The nuclei were counterstained with Hoechst33342 (blue). E: Restoration of DKK1 promoted the β-catenin-dependent transcriptional activity. Tumor cells were transfected with firefly luciferase reporter plasmids containing either wild-type or mutant TCF binding sites (indicated as Wt or Mut on the X axis), and a Renilla luciferase expressing construct. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. The normalized luciferase activity of Vector-transfectants in each experiment was set as relative luciferase activity. ** P < 0.01, compared with Vector-transfectants. F, G: Treatment with β-catenin inhibitor IWP-2 (1 μM) attenuated the pro-migration and pro-invasion effect of DKK1. In (F), after scratching a wound and removing the floating cells, tumors cells without treatment (panel 1, 3) or incubated with 1 μM IWP-2 (panel 2, 4) were applied to the wound scratch assay for 24 h. In (G) tumors cells treated as in (F) were applied to transwell chamber coated with Matrigel and then incubated for 24 h. The data are represented as means ± SEM. *, p <0.05; **, p <0.01.