Figure 3.

miR-135b Activates the Notch1 and Wnt/β-Catenin Pathways

(A) The expression of Notch1, HES1, and β-catenin was significantly increased in CD133- and ALDH1-positive cells compared with CD133- and ALDH1-negative cells, respectively. (B) The expression of HES1 was increased in OS cells that stably expressed miR-135b compared with their corresponding vector control cells. (C) β-Catenin activity was increased in miR-135b-transduced OS cells compared with their corresponding vector control cells. The indicated cells were transfected with TOP or FOP reporter and Renilla pRL-TK plasmid and were subjected to a dual-luciferase assay 48 hr after transfection. (D) The level of β-catenin in the nucleus was increased in cells that stably expressed miR-135b compared with the vector control cells. Nuclear fractions were extracted from the indicated cells, and the indicated proteins were analyzed by western blot. p84 was used as a loading control. (E) The expression of β-catenin and HES1 was assessed by immunofluorescence staining of Saos-2 cells. (F) Immunohistochemistry analysis of HES1 and β-catenin in xenograft tumors that induced by miR-135b transduced Saos-2 cell and vector control cells. (G) The silencing of β-catenin partially inhibited the miR-135b-induced HES1 expression in Saos-2 cells. Saos-2 cells were transfected with the indicated plasmid, and the expression of the proteins shown was measured by western blot 72 hr after transfection. *p < 0.05; **p < 0.01.