RSVL promotes the osteogenic differentiation of BMSCs
To confirm whether RSVL could promote the osteogenic differentiation of BMSCs, we treated them with different concentrations of RSVL (0, 2, 4 and 8 µM) under osteogenic induction for 20 days. First, the structure of RSVL was shown in Fig. 1A. Then, ALP and ARS stainings were applied to detect the roles of RSVL in the osteogenic differentiation of BMSCs. ALP staining showed that, with the increase in the concentration of RSVL, the osteogenic differentiation of BMSCs gradually increased, suggesting that RSVL significantly promoted the osteogenesis of BMSCs in a dose-dependent manner (Fig. 1B). ARS staining and quantification revealed that extracellular matrix mineralization was observably increased by RSVL treatment at 20 days after osteogenic differentiation (Fig. 1C). As shown in supporting Fig. 1D, RSVL treatment significantly upregulated the expressions of osteoblast-related genes, including bone morphogenetic protein (BMP4), Osterix, osteopontin (OPN) and Collagen type I (Col1A), in a dose-dependent manner when added RSVL at concentrations of 2, 4 and 8 µM (Fig. 1D). To further test the potential roles of RSVL in osteogenic differentiation, BMSCs with or without RSVL treatment were cultured in osteogenic induction medium for 20 days, and the protein expression levels of osteogenesis-associated genes, including osteocalcin (OCN) and bone sialoprotein (BSP) were detected by western blot analysis. As shown in Fig. 1E, the protein expression levels of OCN and BSP were found to be increased significantly in cells treated with RSVL (Fig. 1E). Besides, with the increased concentrations of RSVL, the level of OCN and BSP gradually upregulated (Fig. 1E). Taken together, these results demonstrated that RSVL markedly facilitated the osteogenesis of BMSCs in a dose-dependent manner.
miR-320c was downregulated during osteogenic differentiation of BMSCs and under the treatment of RSVL
Previous studies showed that miRNAs played an important role in regulating the osteogenic differentiation of BMSCs. GSE74209, acted as a microRNA array, was applied for the further analysis to detect the expression of dysregulated genes in the Gene Expression Omnibus (GEO) database. As shown in Fig. 2A, miR-320c exhibited the upregulated expression according to the results of the differential analysis (Fig. 2A). The heatmap showed that miR-320c was significantly upregulated in the samples from osteoporotic patients, suggesting that miR-320c might be related to the occurrence and development of bone diseases characterized by bone loss, such as osteoporosis (Fig. 2B). By screening the aberrantly expressed miRNAs during osteogenic differentiation. BMSCs were induced into osteoblasts for 0, 4, 8, 12, 16 and 20 days (Fig. 2C and 2D). Real time qPCR analysis indicated that the expression level of miR-320c decreased along with the osteogenic induction for different time, and the expression level of miR-320c was the lowest after osteogenesis for 20 days, suggesting that miR-320c expression was reduced in a time-dependent manner (Fig. 2E). Then, we subsequently detected the expression level of miR-320c using real time qPCR in RSVL-treated BMSCs. As shown in Fig. 2F, miR-320c expression was reduced in a dose-dependent manner under RSVL treatment, which revealed that miR-320c might participate in the regulation of RSVL-promoted osteogenic differentiation of BMSCs.
The roles of miR-320c in the osteogenic differentiation of BMSCs
To evaluate the effects of miR-320c in the osteogenesis of BMSCs in vivo, the BMSCs transfected with miR-320c mimics or NC were subjected to osteogenic induction for 20 days. ALP staining showed that compared with NC, miR-320c mimics significantly decreased the osteogenic differentiation of BMSCs, suggesting that over-expression of miR-320c in BMSCs could lead to the decreased osteogenic differentiation of BMSCs (Fig. 3A). ARS staining also indicated that transfection of miR-320c mimics markedly suppressed the osteogenic differentiation of BMSCs (Fig. 3B). Further, real time qPCR analysis revealed significantly lower expression of bone matrix genes, including BMP4, Osterix, OPN and Col1A, in miR-320c mimics transfected BMSCs than in NC transfected BMSCs (Fig. 3C). The effects of miR-320c inhibitor were then evaluated in the osteogenic differentiation of BMSCs. Interestingly, ALP and ARS stainings exhibited that the osteogenesis was markedly higher in miR-320c inhibitor group than in NC group, suggesting that knockdown of miR-320c in BMSCs could result in the increased osteogenic differentiation (Fig. 3D and 3E). Real time qPCR analysis revealed the mRNA expression levels of markers of osteogenesis (BMP4, Osterix, OPN and Col1A) were increased in BMSCs after transfection of miR-320c inhibitor (Fig. 3F). These results demonstrate that overexpression of miR-320c inhibited the osteogenic differentiation, while knockdown of miR-320c promoted the osteogenic differentiation of BMSCs.
The effects of RSVL on the adipogenic differentiation of BMSCs and expression of miR-320c during the adipogenic differentiation of BMSCs
To investigate the roles of RSVL in the adipogenic differentiation, we treated BMSCs with RSVL at different concentrations and induced them into adipocytes. ORO staining indicated that the number of oil droplets decreased after treatment of RSVL in a manner-dependent manner, indicating that RSVL might play a negative role in the adipogenesis of BMSCs (Fig. 4A). Besides, compared with the control group, 2, 4 and 8 µM RSVL decreased the mRNA expression levels of adipogenesis-related genes, including leptin, lipoprotein lipase (LPL), adiponectin, and adipocyte protein 2 (αP2) in different degrees (Fig. 4B). As shown in the results of western blot, different concentrations of RSVL significantly downregulated the protein expression levels of peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid binding protein 4 (FABP4), important markers of adipogenesis (Fig. 4C). To further detect the effects of miR-320c on the adipogenic differentiation of BMSCs, the BMSCs were induced into adipocytes under adipogenic differentiation medium. The BMSCs were respectively cultured in adipogenic differentiation medium for 0, 4, 8, 12, 16 and 20 days (Fig. 4D). ORO staining indicated that the number of lipid oil droplets significantly increased with the extension of time (Fig. 4D and 4E). Besides, miR-320c expression was revealed by real time qPCR analysis to gradually increase during adipogenic differentiation of BMSCs, peaking at the adipogenic differentiation of 20 days (Fig. 4F). The above results showed that RSVL significantly inhibited the adipogenic differentiation of BMSCs and miR-320c was upregulated during adipogenesis of BMSCs.
The effects of miR-320c on the adipogenic differentiation of BMSCs
To overexpress or silence miR-320c in BMSCs for functional investigation, we transfected BMSCs with miR-320c mimics, miR-320c inhibitor or NCs and subsequently cultured in adipogenic differentiation medium. As shown in Fig. 5A, ORO staining revealed that overexpression of miR-320c increased the lipid droplet formation in adipogenesis-induced BMSCs, compared with NC (Fig. 5A). Likewise, the mRNA expression levels of four important adipocyte-specific markers, leptin, LPL, adiponectin, and αP2, were also up-regulated compared with NC (Fig. 5B). Conversely, knockdown of miR-320c markedly attenuated the lipid droplet formation of BMSCs under adipogenic differentiation induction, dected by ORO staining (Fig. 5C). In addition, silence of miR-320c suppressed the expression level of adipocyte-related genes, including leptin, LPL, adiponectin, and αP2, of BMSCs after adipogenic differentiation (Fig. 5D).
Overexpression of miR-320c reversed the increased osteogenic differentiation and decreased adipogenic differentiation of BMSCs caused by RSVL
To investigate the functions of miR-320c in RSVL-promoted BMSCs osteogenic differentiation, we used miR-320c mimics to overexpress miR-320c in BMSCs after treatment of 8 µM RSVL. The results of ALP staining showed that overexpression of miR-320c significantly reduced the matrix mineralization deposition and ALP activity, which was upregulated by 8 µM RSVL treatment in BMSCs under osteogenic induction (Fig. 6A). Meanwhile, ARS staining also indicated that in the presence of miR-320c mimics, the osteogenic differentiation increased by 8 µM RSVL treatment was significantly decreased (Fig. 6B). Besides, the upregulated mRNA expression level of osteoclast-specific genes in the RSVL-treated BMSCs were significantly down-regulated, including leptin, LPL, adiponectin, and αP2 (Fig. 6C). To further investigate the role of miR-320c in RSVL-suppressed BMSCs adipogenic differentiation, BMSCs were transfected with miR-320c mimics and treated with RSVL under adipogenic differentiation medium. ORO staining revealed that the number of oil droplets were decreased by the RSVL treatment, which could be increased by overexpression of miR-320c (Fig. 6D). Real time qPCR analysis indicated that the mRNA expression level of adipocyte-specific genes, including leptin, LPL, adiponectin, and αP2, was reduced by RSVL treatment and then downturned by miR-320c mimics (Fig. 6E). The above data suggested that miR-320c played a negative role in RSVL-promoted osteogenic differentiation and RSVL-suppressed adipogenic differentiation of BMSCs.
MiR-320c regulates RSVL-promoted osteogenic differentiation of BMSCs by targeting Runx2
To confirm the possible targets of miR-320c in RSVL-promoted BMSCs osteogenic differentiation, two miRNA target prediction systems (TargetScan Human 7.2) were used in our study to look for miR-320c-targeting mRNAs. Among the putative mRNAs, we noticed that Runx2 had close relationship with miR-320c and the binding sites between miR-320c and Runx2 were predicted using miRanda and TargetScan (Fig. 7A). Based on these data, we speculated that Runx2 was the directly target of miR-320c. Luciferase reporter assay was performed to verify the hypothesis. Luciferase reporter assay showed that the relative luciferase activity of Runx2-3'UTR-WT was significantly repressed duo to miR-320c mimics’ addition when compared with control (Fig. 7B). In contrast, the predicted binding sequence mutation completely abrogated this repression (Fig. 7B). Additionally, real time qPCR and western blot analysis revealed that overexpression of miR-320c could decrease the Runx2 expression, while knockdown of miR-320c could increase the Runx2 expression in BMSCs (Fig. 7C-7E). Taken together, these results confirm that Runx2 was the directly target of miR-320c in RSVL-promoted osteogenic differentiation of BMSCs.