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  • The important role of miRNAs in cellular differentiation has


    The important role of miRNAs in cellular differentiation has been proposed recently. Previous studies had suggested roles for different miRNAs during osteogenesis [20]. Certain miRNAs could promote osteoblast differentiation, such as miR-194 [20], whereas others could inhibit the cell differentiation of osteogenesis-related cells, including miR-155 [26]. As a member of the miR-15 family, miR-195 was also reported to play a key role in many cellular pathways. A study by Zhang et al. found that the up-regulation of miR-195 accelerated oxidative stress-induced retinal endothelial cell injury by targeting mitofusin 2 in diabetic rats [27], indicating its promoting role in oxidative stress damage. A decrease of miR-195 promoted chondrocyte proliferation and the maintenance of chondrogenic phenotype by targeting the FGF-18 pathway [28]. Grünhagen et al. demonstrated that the miR-497–miR-195 cluster expression level was reduced during osteogenesis of primary human osteoblasts and that miR-195 alters the gene regulatory network of osteoblast differentiation and impairs the induction of BMP responsive genes [16]. These reports indicated that miR-195 has multiple biological functions in addition to osteoblast differentiation. Previous studies have shown aberrant miR-195 expression in various types of human cancer, such as gastric, Wortmannin and breast cancer [29], [30]. In breast cancer, Li et al. found that miR-195 and miR-497 are down-regulated in human breast cancer tissues and cell lines and inhibit colon formation by directly targeting RAF-1 [31], which suggested a possible mechanism of miR-195 inhibition on osteoblast differentiation. We demonstrated that miR-195 over-expression hampered the cellular responses to BMP-2 in MC3T3-E1 cells. The viability of MC3T3-E1 cells was significantly decreased due to the inhibition of miR-195 over-expression (Fig. 3, C). Furthermore, under BMP-2 inducing conditions, miR-195 over-expression resulted in the limited phosphorylation levels of ERK, MEK and Smad1/5 (Fig. 4, E), which dramatically suppressed the osteoblast differentiation of MC3T3-E1 cells, primarily manifesting in the decreased ALP activity and down-regulated gene expression levels of osteogenic markers (Fig. 4, A–D). Our findings for the first time demonstrated the inhibition effect of miR-195 on osteoblast differentiation and were in accordance with the previous study [16]. Although it was reported that miR-195 could target the RAF-1 3′-UTR in thyroid cancer cells, whether there is similar negative regulation in osteoblast differentiation remains unclear. In this study, RAF-1 was observed to be the target of miR-195 in the osteogenesis of MC3T3-E1 cells. Transfection with miR-195 effectively reduced RAF-1 transcription and suppressed the RAF-1 (or RAF-1L613V) –amplified osteoblast differentiation of MC3T3-E1 cells induced by BMP-2 (Fig. 4, Fig. 5). The positive regulatory effect of RAF-1 (or RAF-1L613V) on osteogenic gene expression (Runx2, OSX, ALP, OCN, and DLX5) was also reversed (Figs. 4C, D and 5E). In addition, our findings showed that miR-195 transfection significantly limited the BMP-2 signalling pathway. In a previous study, Grünhagen et al. observed that miR-195 inhibited the BMP-2 signalling pathway by targeting several targets including Smad5, Tgfbr3, Furin [16], which was in keeping with the results of our study. We confirmed that miR-195 decreased the mRNA and protein expression of RAF-1 by directly targeting the 3′-UTR of RAF-1 mRNA; it is suggested that miR-195 could suppress osteoblast differentiation by targeting RAF-1 in addition to the canonical BMP-2/Smad pathway. Taken together, our results indicated that RAF-1 and RAF-1L613V exert an amplified effect with BMP-2 on osteoblast differentiationin in MC3T3-E1 cells, whereas miR-195 inhibited both normal and abnormal activation of osteoblast differentiation in the cells via targeting RAF-1. miR-195 might be a potent therapeutic agent for the prevention and treatment of L613V-induced bone deformity in Noonan syndrome.