Figure 6.

HBx of HBV activates the PI3K/AKT pathway to promote cell migration and invasion via miR-520c-3p-mediated downregulation of PTEN

(A) Both miR-520c-3p and HBV/HBx activate AKT. Huh7 cells were transfected with HBV or HBx (left), miR-520c-3p mimics/inhibitors or corresponding NC miRNA (right). Huh7 cells were transfected with HBV/HBx or miR-520c-3p, after 48 h transfection, the cells were analyzed for the levels of AKT, p-AKT, Vimentin, E-cadherin, MMP9, and PTEN levels by western blot. (B) Downregulation of PTEN is required for the activation of AKT by miR-520c-3p. Huh7 cells were co-transfected with pEF-Flag (control) or PTEN plus miR-520c-3p mimic, and western blot analyses were performed for the indicated proteins. (C) Inhibiting miR-520c-3p prevents the activation of AKT by HBx, while knocking down PTEN reverses this effect. Huh7 cells were co-transfected with HBx, miR-520c-3p inhibitor, and siPTEN and western blot analyses were performed for the indicated proteins. (D) AKT activation is required for miR-520c-3p-induced cell migration and invasion. Huh7 cells were transfected with miR-520c-3p mimic or NC-mimic for 36 h, and treated with MK2206, a specific inhibitor of AKT phosphorylation, or dimethyl sulfoxide for 12 h. The cells were then subjected to western blot analyses for the indicated proteins (left) or cell migration and invasion assays as in Figure 2A (right). (E) AKT activation is required for HBx-induced cell migration and invasion. Huh7 cells at 36 h after transfection with pxj40-HBx-HA or empty vector were treated with MK2206 or dimethyl sulfoxide for 12 h. The cells were then subjected to western blot analyses for the indicated proteins (left) or cell migration and invasion assays as in Figure 2A (right). Three independent experiments were performed, and representative data are shown. ∗∗p < 0.01, ∗∗∗p < 0.001 in (D–E).