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    • The important role of miRNAs in cellular differentiation has

    2019-11-30

    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.