MiRNA-10b Reciprocally Stimulates Osteogenesis and Inhibits Adipogenesis Partly through the TGF-β/SMAD2 Signaling Pathway
Li Hongling1, Fan Junfen1, Fan Linyuan1, Li Tangping1, Yang Yanlei1, Xu Haoying1, Deng Luchan1, Li Jing1, Li Tao2,3, Weng Xisheng2, Wang Shihua1,*, Chunhua Zhao Robert1,*
1Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Center of Excellence in Tissue Engineering Chinese Academy of Medical Sciences, Beijing Key Laboratory (No. BZO381), Beijing 100005, China. 2Department of Orthopaedic Surgery, Peking Union Medical College Hospital, Peking Union Medical College, Beijing 100730, China. 3Current address: Department of Orthopaedic Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266003, China.
As the population ages, the medical and socioeconomic impact of age-related bone disorders will further increase. An imbalance between osteogenesis and adipogenesis of mesenchymal stem cells (MSCs) can lead to various bone and metabolic diseases such as osteoporosis. Thus, understanding the molecular mechanisms underlying MSC osteogenic and adipogenic differentiation is important for the discovery of novel therapeutic paradigms for these diseases. miR-10b has been widely reported in tumorigenesis, cancer invasion and metastasis. However, the effects and potential mechanisms of miR-10b in the regulation of MSC adipogenic and osteogenic differentiation have not been explored. In this study, we found that the expression of miR-10b was positively correlated with bone formation marker genes ALP, RUNX2 and OPN, and negatively correlated with adipogenic markers CEBPα, PPARγ and AP2 in clinical osteoporosis samples. Overexpression of miR-10b enhanced osteogenic differentiation and inhibited adipogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) in vitro, whereas downregulation of miR-10b reversed these effects. Furthermore, miR-10b promoted ectopic bone formation in vivo. Target prediction and dual luciferase reporter assays identified SMAD2 as a potential target of miR-10b. Silencing endogenous SMAD2 expression in hADSCs enhanced osteogenesis but repressed adipogenesis. Pathway analysis indicated that miR-10b promotes osteogenic differentiation and bone formation via the TGF-β signaling pathway, while suppressing adipogenic differentiation may be primarily mediated by other pathways. Taken together, our findings imply that miR-10b acts as a critical regulator for balancing osteogenic and adipogenic differentiation of hADSCs by repressing SMAD2 and partly through the TGF-β pathway. Our study suggests that miR-10b is a novel target for controlling bone and metabolic diseases.
Li Hongling,Fan Junfen,Fan Linyuan, et al. MiRNA-10b Reciprocally Stimulates Osteogenesis and Inhibits Adipogenesis Partly through the TGF-β/SMAD2 Signaling Pathway[J]. Aging and disease,
2018, 9(6): 1058-1073.
Figure 1. Expression pattern of miR-10b. (A) The dynamic expression of miR-10b during osteogenic and adipogenic differentiation of hADSCs. (B) The correlation between the expression of osteogenic-related genes and miR-10b in clinical osteoporosis samples. (C) The conservation of miR-10b in mammals. (D) miR-10b expression levels in various cell lines and mouse tissues (M-osteoblast: hADSC-derived osteoblast) were detected by qRT-PCR. The data, normalized to GAPDH or U6, are averages of 3 independent experiments (mean ± SD).
Figure 2. MiR-10b promotes osteogenic differentiation and inhibits adipogenic differentiation of hADSCs. (A) The miR-10b-expressing lentivirus increased the expression of mature miR-10b in hADSCs, analyzed by stem-loop qRT-PCR. (B and C) ALP staining was performed on day 4 and ALP activity was detected during osteogenic differentiation. (D) Alizarin red staining was performed to indicate mineral deposition on day 12. (E and F) Lenti-10b increased the mRNA and protein expression levels of osteogenic-specific markers on day 6 of osteogenic differentiation. (G) Oil red O staining was performed to detect the lipid droplets formation on day 10 of adipogenic differentiation. (H) The dye of oil red O-positive cells was extracted by isopropanol, and the OD value was quantified at 510 nm wavelength. (I and J) Lenti-10b decreased the mRNA and protein expression levels of adipogenic-specific markers. The data, normalized to U6 or GAPDH, are averages of 3 independent experiments (mean ± SD). *P<0.05; **P<0.01; ***P<0.001 compared with the control. Scale bars: 200 μm.
Figure 3. MiR-10b promotes the ectopic bone formation of hADSCs in vivo. (A) H&E staining was used to analyze osteoid formation in the xenografts. (B) Masson trichrome staining indicated collagen. (C) Safranin O staining revealed cartilage formation in the xenografts. (D) Immunohistochemical staining showed the expression levels of osteogenic markers in the xenografts. Black arrows represent positive signals. Scale bars: 200 μm.
Figure 4. Downregulation of endogenous miR-10b suppresses osteogenic differentiation and enhances adipogenic differentiation. (A) miR-10b expression was determined by stem-loop RT-PCR. (B and C) ALP staining and ALP activity were performed after inhibiting miR-10b. (D) Alizarin red staining was performed on day 12. (E and F) qRT-PCR and western blot were performed to analyze the mRNA and protein levels of osteogenic-specific markers after miR-10b inhibitor transfection. (G and H) Oil red O staining and extraction were performed to detect the formation of lipid droplets on day 10 of adipogenic differentiation. (I and J) The expression of adipogenic-specific markers was analyzed by qRT-PCR and western blot. The data, normalized to GAPDH, are averages of 3 independent experiments (mean ± SD). *P<0.05; **P<0.01; ***P<0.001 compared with the control. Scale bars: 200 μm.
Figure 5. SMAD2 is a direct target of miR-10b. (A and B) Bioinformatic analysis was performed to predict the binding seed sequence of miR-10b with the 3’UTR of SMAD2. The wild-type (WT) or mutant-type (MUT) constructs were inserted into the psiCHECK-2 reporter vector. Luciferase activity was measured in the lysates, and the values were normalized to the psiCHECK-2 vector. (C) Western blot and qRT-PCR analyzed the expression of SMAD2 in hADSCs. (D) The knockdown efficiency of three SMAD2 siRNAs was confirmed by qRT-PCR and western blot. (E) ALP staining and activity assay were performed to analyze ALP expression and activity on day 6. (F) Alizarin red staining showed increased calcification after SMAD2 knockdown. (G and H) The critical regulators of osteogenesis were analyzed by qRT-PCR and western blot. (I) Oil red O staining and extraction were performed to detect the formation of lipid droplets on day 10 of adipogenic differentiation. (J and K) qRT-PCR and western blot were used to analyze adipogenic-specific markers after SMAD2 knockdown. The data, normalized to GAPDH, are averages of 3 independent experiments (mean ± SD). *P<0.05; **P<0.01 compared with the control. Scale bars: 200 μm.
Figure 6. SMAD2 knockdown reverses the effect of miR-10bI on osteogenic and adipogenic differentiation of hADSCs. (A and B) ALP staining and activity were performed on day 6 of osteogenic differentiation. (C and D) Western blot and qRT-PCR were used to analyze osteogenic factor expression after different treatments. (E and F) Oil red O staining and extraction were performed to detect the lipid droplets formation on day 10 of adipogenic differentiation. (G and H) Western blot and qRT-PCR were performed to analyze protein and mRNA expression levels of adipogenic markers, respectively. The data, normalized to GAPDH, are averages of 3 independent experiments (mean ± SD). *P<0.05; **P<0.01 compared with the control. Scale bars: 200 μm.
Figure 7. MiR-10b regulates hADSC differentiation partly through the TGF-β signaling pathway. (A) ALP staining was used to analyze the effects of different concentrations of TGF-β1 on ALP expression. (B) Oil red O staining was performed to analyze the effects of different concentrations of TGF-β1 on the formation of lipid droplets. (C) ALP staining and activity were detected on day 6 of osteogenic differentiation. Oil red O staining and extraction were analyzed on day 10 of adipogenic differentiation after different treatments. (D) The mRNA levels of osteogenic- and adipogenic-related genes after different treatments. (E) Western blot analyzed the protein levels of the osteogenic and adipogenic factors, and TGF-β signaling pathway-related molecular elements after different treatments. The data, normalized to GAPDH are averages of 3 independent experiments (mean±SD). *P<0.05; **P<0.01; ***P<0.001 compared with the control. Scale bars: 200 μm.
Figure 8. A schematic model illustrating that miR-10b mediated function in osteogenic and adipogenic differentiation of hADSCs. miR-10b suppresses SMAD2 expression at the post-transcriptional level, resulting in downregulation of the TGF-β signaling pathway, and thereby promoting osteogenic differentiation and decreasing adipogenic differentiation.
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