miR-27a attenuates adipogenesis and promotes osteogenesis in steroid-induced rat BMSCs by targeting PPARγ and GREM1

Abstract

The imbalance between adipogenic and osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs) plays a significant role in the pathogenesis of steroid-induced osteonecrosis of the femoral head (ONFH). Several microRNAs (miRNAs) are involved in regulating adipogenesis and osteogenesis. In this study, we established a steroid-induced ONFH rat model to identify the potential relevant miRNAs. We identified 9 up-regulated and 28 down-regulated miRNAs in the ONFH rat model. Of these, miR-27a was down-regulated and negatively correlated with peroxisome proliferator-activated receptor gamma (PPARγ) and gremlin 1 (GREM1) expression. Further studies confirmed that PPARγ and GREM1 were direct targets of miRNA-27a. Additionally, adipogenic differentiation was enhanced by miR-27a down-regulation, whereas miRNA-27a up-regulation attenuated adipogenesis and promoted osteogenesis in steroid-induced rat BMSCs. Moreover, miRNA-27a up-regulation had a stronger effect on adipogenic and osteogenic differentiation in steroid-induced rat BMSCs than si-PPARγ and si-GREM1. In conclusion, we identified 37 differentially expressed miRNAs in the steroid-induced ONFH model, of which miR-27a was down-regulated. Our results showed that miR-27a up-regulation could inhibit adipogenesis and promote osteogenesis by directly targeting PPARγ and GREM1. Thus, miR-27a is likely a key regulator of adipogenesis in steroid-induced BMSCs and a potential therapeutic target for ONFH treatment.

Figure 1. miR-27a was down-regulated while PPARγ and GREM1 were up-regulated in the steroid-induced ONFH rat model.

(a) HE staining showed that the bone trabecula was thinner, sparser, smaller, and more commonly fractured and disordered in the Model group compared with the Control group. (b) The microarray analysis identified 37 differentially expressed miRNAs in the Model group. (c) qRT-PCR detected a significant down-regulation of miR-27a in the Model group. (d) qRT-PCR detected the up-regulation of miR-182 in the Model group. (e, f) qRT-PCR detected a significant up-regulation of the PPARγ and GREM1 mRNAs in the Model group. (g, h) The PPARγ and GREM1 mRNA levels were negatively correlated with miR-27a expression. ^*P < 0.05.

Figure 2. PPARγ and GREM1 were direct targets of miRNA-27a.

(a) PPARγ and GREM1 share a matching 3′UTR sequence that targets the seed region of miR-27a. The sequences are presented. (b) Western blots showed the down-regulation of PPARγ and GREM1 in the miR-27a mimics group and up-regulation of PPARγ and GREM1 in the miR-27a inhibitor group compared with the mimics or inhibitor control groups. (c, d) The dual luciferase assay showed significantly reduced luciferase activity in BMSCs that were co-transfected with the miR-27a mimics and pmirGLO-wt-PPARγ compared with cells co-transfected with miR-27a mimics and pmirGLO-mt-PPARγ, and significantly enhanced luciferase activity was observed in BMSCs that were co-transfected with miR-27a inhibitor and pmirGLO-wt-PPARγ compared with the cells that were co-transfected with the miR-27a inhibitor and pmirGLO-mt-PPARγ. A similar result was observed in BMSCs that were co-transfected with the miR-27a mimics and pmirGLO-wt-GREM1 or the cells that were co-transfected with the miR-27a inhibitor and pmirGLO-wt-GREM1. ^*P < 0.05.

Figure 3. Adipogenic differentiation was enhanced, osteogenic differentiation was inhibited and miR-27a was down-regulated in steroid-induced rat BMSCs.

(a) Oil red O staining detected more lipid droplets in BMSCs from the Model group compared with the Control group on day 14. (b) A significant increase in the TG content of BMSCs from the Model group was observed in comparison with the Control group on days 7 and 14. (c,d) Significant decreases in the ALP and OST contents in the Model group were observed in comparison with the Control group. (e,f,i) qRT-PCR showed that PPARγ, GREM1, and C/EBPα were significantly up-regulated in BMSCs from the Model group compared with the Control group. (g,h,j) qRT-PCR showed that Runx2, Bmp-2 and miR-27a were significantly down-regulated in BMSCs from the Model group compared with the Control group. (k) The western blotting results were consistent with the qRT-PCR results: PPARγ, GREM1, and C/EBPα were significantly up-regulated while Runx2 and Bmp-2 were significantly down-regulated in BMSCs from the Model group. *P < 0.05.

Figure 4. miRNA-27a up-regulation attenuated adipogenic differentiation and promoted osteogenic differentiation in steroid-induced rat BMSCs.

(a) The qRT-PCR conducted at 24 h post-transfection showed that miR-27a expression was significantly increased in the miR-27a group compared with the Blank group. (b) Oil red O staining revealed fewer lipid droplets in BMSCs from the miR-27a group compared with the Model and Scramble groups. (c) ALP staining showed that a greater number of BMSCs were stained blue/purple in the miR-27a group than in the Model and Scramble groups. (d) A significantly reduction in the TG content was observed in the miR-27a group. (e,f) Significant increases in the ALP content of BMSCs and the OST content in the culture medium were observed in the miR-27a group. (g) The qRT-PCR results showed a significant reduction in the expression of adipogenesis-associated genes (PPARγ and C/EBPα). Furthermore, osteogenesis-associated genes were significantly and differentially expressed in BMSCs from the miR-27a group: GREM1 was down-regulated, whereas Runx2 and Bmp-2 were up-regulated. (h) The western blotting results were similar to the qRT-PCR results. *P < 0.05.

Figure 5. The expression of PPARγ and GREM1 without 3′UTRs rescued the effects of miRNA-27a overexpression on adipogenic and osteogenic differentiation in steroid-induced rat BMSCs.

(a,b) Western blots showed the up-regulation of PPARγ and GREM1 in groups that were co-transfected with pcDNA3.1-PPARγ or pcDNA3.1-GREM1 (without 3′UTRs). (c) Higher TG levels were observed in steroid-treated BMSCs that were co-transfected with pcDNA3.1-PPARγ and miR-27a compared to the group that was transfected with miR-27a alone. (d) Lower ALP levels were observed in steroid-treated BMSCs that were co-transfected with pcDNA3.1-GREM1 and miR-27a compared to the group that was transfected with miR-27a alone. *P < 0.05.

Figure 6. miRNA-27a up-regulation had a stronger effect on attenuating adipogenic differentiation in steroid-induced rat BMSCs than si-PPARγ and si-GREM1.

(a) The qRT-PCR conducted at 24 h post-transfection demonstrated successful transfection. (b) Reduced PPARγ expression was observed in the miR-27a and si-PPARγ groups compared with the Model and si-GREM1 groups, and reduced expression of C/EBPα was observed in the miR-27a group. Furthermore, GREM1 was significantly down-regulated in the miR-27a and si-GREM1 groups, whereas Runx2 and Bmp-2 were significantly up-regulated in the miR-27a group compared with the Model, si-PPARγ and si-GREM1 groups. (c) The western blotting results were in accordance with the qRT-PCR results. (d) ALP staining revealed an increased number of BMSCs that were stained blue/purple in the miR-27a group than in the other groups. (e) Oil red O staining showed significantly fewer droplets in BMSCs from the miR-27a group compared with the other groups. (f) A significantly reduction in TG contents was observed in BMSCs from the miR-27a group compared with the Model, si-GREM1 and si-PPARγ groups. (g,h) Significant increases in ALP and OST content were observed in the miR-27a group compared with the Model, si-PPARγ and si-GREM1 groups. *P < 0.05.