As small non coding RNAs microRNAs miRNAs

As small non-coding RNAs, microRNAs (miRNAs) can control gene expression at the post-transcriptional level by binding to the 3′-untranslated regions (3′-UTR) of messenger RNAs (mRNA) [15], [16]. miRNAs have been anticipated to regulate virtually all cellular mechanisms [17] and to play an important role in cellular responses, including cell proliferation, apoptosis and differentiation [18], [19]. An increasing number of miRNAs have recently been identified to strongly regulate the osteoblast differentiation and bone formation, such as microRNA-194 (miR-194) [20] and microRNA-195 (miR-195) [15], [16]. These findings suggest that it is feasible to develop methods for improving osteoblast differentiation by targeting critical miRNAs. Recent reports have demonstrated that the exogenous overexpression of miR-195 significantly inhibits the protein expression of RAF-1 and blocks thyroid cancer cell proliferation [21]. The expression of miR-195 was inversely associated with RAF-1 expression in breast cancer cell lines and tissue specimens [22]. Furthermore, the expression of miR-195 or knockdown of RAF-1 can similarly reduce tumour cell survival but increase apoptosis through the down-regulation of RAF-1 [22]. Therefore, it could be seen that RAF-1 is the target gene of miR-195. Whether miR-195 could inhibit osteoblast differentiation of MC3T3-E1 N-3-oxo-dodecanoyl-L-Homoserine lactone abnormally activated by RAF-1 or RAF-1L613V, however, remains largely unknown. In this study, we first identified that RAF-1, especially RAF-1L613V, has an amplification effect on osteoblast differentiation of MC3T3-E1 cells induced by BMP-2. Next, the inhibitory role of miR-195 in regulating cell activity and differentiation of MC3T3-E1 cells was also confirmed. Further study showed that miR-195 largely suppressed the wild-type RAF-1 (WT) or RAF-1L613V (L613V)–induced viability and osteoblast differentiation of MC3T3-E1 cells by targeting RAF-1 3′-UTR. Our findings would provide a novel therapeutic agent for the treatment of RAF-1 or RAF-1L613V–induced bone deformity in Noonan syndrome.
Materials and methods
Results
Discussion The bone deformity in Noonan syndrome is different from any other diseases in the skeletal system. This deformity is caused by dysfunction of the RAS/MAPK signalling pathway [6]. RAF is a mitogen-activated protein kinase that is central in the conserved RAS/MAPK signalling pathway and relays signals from activated RAS proteins via MAPK/ERK kinase 1/2 (MEK1/2) to ERK1/2, the key effectors of this pathway [23]. Constitutive activation of this pathway as a result of mutations is considered a key event in the development of Noonan syndrome [24]. As an antiapoptotic factor, RAF-1 is an important member of the RAF proto-oncogene family [6]. Studies have confirmed that RAF-1 had many connections with cell proliferation and osteoblast differentiation [9], [10]. RAF-1 could decrease the number of apoptotic osteocytes, thereby promoting cell proliferation and differentiation of MC3T3-E1 cells to a certain extent [10]. L613V, a mutant of RAF-1, is a major risk factor for heart failure and death in Noonan syndrome. RAF-1 L613V confers increased kinase activity and is highly associated with the activation of MEK1/2-ERK1/2 mitogen-activated protein kinases [12]. Treatment of these mice with MEK inhibitors attenuated many phenotypic abnormalities, including bone deformity [12]. In the present study, our findings provided evidence that RAF-1, especially RAF-1L613V, amplified BMP-2 induction and abnormally activated proliferation and osteoblast differentiation of MC3T3-E1 cells. The addition of WT RAF-1 or RAF-1L613V amplified the genes expression of osteogenic markers (Runx2, OSX, ALP, OCN, and DLX5) (Figs. 2D and 5E). However, the promotion of RAF-1 on osteoblast differentiation was not evident in the absence of BMP-2 treatment. To clarify the detailed mechanisms, further investigations showed that the synergistic effect of L613V and BMP-2 significantly enhanced the phosphorylation levels of ERK, MEK and Smad1/5 (Figs. 2E and 4E). The results described above suggested that L613V contributed to the activation of MEK1/2-ERK1/2 mitogen-activated protein kinases, thereby up-regulating the phosphorylation levels of downstream Smad1/5 to significantly increase the gene expression of osteogenic markers (Runx2, OSX, ALP, OCN, and DLX5) (Figs. 2D and 5E). This signalling pathway could be simply expressed as WT (L613V)/MEK1/2/ERK1/2/Smad1/5/Runx2. Although most previous reports showed that the ERK pathway does not directly activate Smad1/5, the ERK pathway might have an effect on Smad1/5 via an indirect manner because this pathway could regulate numerous downstream molecules and pathways. Additionally, our finding was supported by previous studies. For example, an article published in Experimental & Molecular Medicine demonstrated that using the ERK inhibitor PD98059 could inhibit fucoidan-induced phosphorylation of Smad1/5 [25] which is, to a certain extent, consistent with our results. However, the detailed mechanism warrants further investigation in a future study. As an important downstream signalling molecule of the BMP-2 signalling pathway, p38 was not affected by WT or L613V. Although there was no significant difference in most cases between WT and L613V, evidence showed that L613V appeared to have a more powerful effect on osteoblast differentiation.