Figure 3. Direct inhibition of the C-Met’s protein’s translation by miR-198 and -206 modulates the downstream Akt and the ERK ½ signaling pathways.

The expression of C-Met was assessed at mRNA level by RT-qPCR (A) and at protein level by Western blotting (B) in the HOS LucF-GFP Osteosarcoma cell line pre-miRNAs’ (left panel) or anti-miRNAs’ transfections (right panel). Error bars show s.d. for n = 3 measurements from representative experiments. GAPDH and B2M were used as housekeeping genes. (C) The expression of Akt, P-Akt, ERK1/2 and P-ERK1/2 was assessed by Western blotting in the HOS LucF-GFP Osteosarcoma cell line 48 hours after either the pre-miRNAs’ transfections (left panel) or the anti-miRNAs’ ones (right panel). For all the Western blots presented, the GAPDH was used as an internal loading control. (D) The HOS LucF-GFP Osteosarcoma cells were co-transfected with the indicated pre-miRNAs (left panel) or anti-miRNAs (right panel), together with either the UTR-reporter (Psi-check2 C-Met 3′UTR, white bars) or the control vector (Psi-check2 control, black bars), the cells were lysed forty-eight hours after transfections and the luciferase bioluminescence was assessed. Results are shown as relative luciferase units (RLU) normalized to the control pre/anti-miR, and the control vector’s results were assigned to 1. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Error bars show s.d. for n = 3 measurements from representative experiments. One-way ANOVA and Dunnett’s multiple comparison tests were used to compare the significance of the results.