Recent reports have demonstrated that RAF-1L613V (a mutant of RAF-1) mutant mice show bone deformities similar to Noonan syndrome. It has been suggested that RAF-1L613V might abnormally activate osteoblast differentiation of MC3T3-E1 cells.Methods:
To demonstrate that RAF-1 is associated with bone deformity and that RAF-1L613V dependent bone deformity could be inhibited by microRNA-195 (miR-195), we first investigated the amplifying influence of wild-type RAF-1 (WT) or RAF-1L613V (L613V) on the viability and differentiation of MC3T3-E1 cells induced by bone morphogenetic protein-2 (BMP-2) via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Subsequently, we investigated the blocking effect and its mechanism of miR-195 for abnormal activation of osteoblast differentiation of MC3T3-E1 cells via targeting RAF-1.Results:
RAF-1, especially RAF-1L613V, abnormally activates osteoblast differentiation of MC3T3-E1 cells induced by BMP-2. Meanwhile, miR-195 could inhibit the cell viability and differentiation of MC3T3-E1 cells. Transfection of miR-195 largely suppressed the L613V-induced viability and osteoblast differentiation of MC3T3-E1 cells and attenuated the accelerative effect of L613V on runt-related transcription factor-2 (Runx2), Osterix (OSX), alkaline phosphatase (ALP), osteocalcin (OCN), and distal-less homeobox 5 (DLX5) osteogenic gene expressions. In addition, miR-195 decreased the expression of RAF-1 mRNA and protein by directly targeting the 3′-untranslated regions (3′-UTR) of RAF-1 mRNA in MC3T3-E1 cells.Conclusions:
Our findings indicated that miR-195 inhibited WT and L613V RAF-1 induced hyperactive osteoblast differentiation in MC3T3-E1 cells by targeting RAF-1. miR-195 might be a novel therapeutic agent for the treatment of L613V-induced bone deformity in Noonan syndrome.