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. 2015 Aug 6;6(8):e1851.
doi: 10.1038/cddis.2015.221.

MiR-26a functions oppositely in osteogenic differentiation of BMSCs and ADSCs depending on distinct activation and roles of Wnt and BMP signaling pathway

X Su  1 , L Liao  2 , Y Shuai  2 , H Jing  2 , S Liu  2 , H Zhou  3 , Y Liu  4 , Y Jin  5
Affiliations

MiR-26a functions oppositely in osteogenic differentiation of BMSCs and ADSCs depending on distinct activation and roles of Wnt and BMP signaling pathway

X Su et al. Cell Death Dis. .

Abstract

MicroRNAs (miRNAs) emerge as important regulators of stem cell lineage commitment and bone development. MiRNA-26a (miR-26a) is one of the important miRNAs regulating osteogenic differentiation of both bone marrow-derived mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (ADSCs). However, miR-26a functions oppositely in osteogenic differentiation of BMSCs and ADSCs, suggesting distinct post-transcriptional regulation of tissue-specific MSC differentiation. However, the molecular basis is largely unknown. Here, we report that the function of miR-26a is largely depended on the intrinsic signaling regulation network of MSCs. Using bioinformatics and functional assay, we confirmed that miR-26a potentially targeted on GSK3β and Smad1 to regulate Wnt and BMP signaling pathway. Overall comparative analysis revealed that Wnt signaling was enhanced more potently and played a more important role than BMP signaling in osteogenic differentiation of BMSCs, whereas BMP pathway was more essential for promoting osteogenic differentiation of ADSCs. The distinct activation pattern and role of signaling pathways determined that miR-26a majorly targeted on GSK3β to activate Wnt signaling for promoting osteogenic differentiation of BMSCs, whereas it inhibited Smad1 to suppress BMP signaling for interfering with the osteogenic differentiation of ADSCs. Taken together, our study demonstrated that BMSCs and ADSCs applied different signaling pathway to facilitate their osteogenic differentiation, which determined the inverse function of miR-26a. The distinct transcriptional regulation and post-transcriptional regulation network suggested the intrinsic molecular differences between tissue-specific MSCs and the complexity in MSC research and MSC-based cell therapy.

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Figures

Figure 1
Figure 1
MiR-26a inversely affects the osteogenic differentiation of BMSCs and ADSCs. (a and b) Expression of miR-26a in BMSCs (a) and ADSCs (b) during osteogenic induction. (c–h) BMSCs (c–e) and ADSCs (f–h) were transfected with miR-26a precursors (pre-miR-26a), miR-26a inhibitors (anti-miR-26a) and negative control (miR-cont) for 48 h before osteogenic induction. ALP staining and alizarin red staining were performed after 7 days or 14 days of induction separately (c and f). Alizarin red staining was extracted with cetylpyridinium chloride and quantified by spectrophotometer (d and g). Expression of Alp, Runx2 and Ocn (normalized to β-actin) was determined by real-time RT-PCR (e and h). (i) BMSCs and ADSCs transfected with miR-26a precursors, miR-26a inhibitors and negative control in vitro were transplanted with HA-TCP subcutaneously into immunocompromised mice for 8 weeks. The transplants were harvested and stained with H&E. B, bone; TCP, hydroxyaptite-tricalcium phosphate. (j) Osteoid formation in transplants was evaluated as osteoid area per total area in H&E staining photos with Image Pro software. Scale bar: 200 μm. Results are shown as mean±S.D. *P<0.05, **P<0.01, ***P<0.001 (n=4)
Figure 2
Figure 2
MiR-26a targets on both Smad1 and GSK3β mRNA. (a) The diagram showed the binding region of miR-26a to 3′ UTR of Smad1 by complementary base paring. (b) Luciferase activity of pMIR-Reporter containing Smad1 3′ UTR was measured 48 h after co-transfection with pre-miR-26a, anti-miR-26a or miR-control. (c) The diagram showed the binding region of miR-26a to 3′ UTR of GSK3β. (d) Luciferase activity of pMIR-Reporter containing GSK3β 3′ UTR was measured 48 h after co-transfection. (e–h) BMSCs and ADSCs were transfected with pre-miR-26a, anti-miR-26a or miR-control for 48 h. Smad1 and GSK3β mRNA level in BMSCs (e) and ADSCs (g) was determined by real-time RT-PCR. Smad1 and GSK3β protein accumulation in BMSCs (f) and ADSCs (h) was determined by western blot. Relative protein abundance of each blots was normalized to the gray value of β-actin. Results are represented as mean±S.D. *P<0.05, **P<0.01, ***P<0.001 (n=3)
Figure 3
Figure 3
MiR-26a focuses on different targets to control BMSC and ADSC osteogenic differentiation. Smad1 or GSK3β siRNA was co-transfected with negative control, pre-miR-26a and anti-miR-26a into BMSCs (a–c) or ADSCs (d–f) for 48 h before osteogenic induction. (a, b, d and e) ALP staining and alizarin red staining were performed after osteogenic induction. (c and f) Alp and Ocn mRNA expression was analyzed by real-time RT-PCR after induction. Results are shown as mean±S.D. *P<0.05, **P<0.01, ***P<0.001 (n=3)
Figure 4
Figure 4
GSK3β and Smad1 have distinct importance in osteogenic differentiation of BMSCs and ADSCs. (a, b, d and e) ALP staining and alizarin red staining of BMSCs (a and b) and ADSCs (d and e) transfected with GSK3β siRNA, Smad1 siRNA or negative control after osteogenic induction. (c and f) Alp, Ocn and Runx2 mRNA expression was determined by real-time RT-PCR. Results are shown as mean±S.D. *P<0.05, **P<0.01, ***P<0.001 (n=3)
Figure 5
Figure 5
Wnt and BMP signaling are differentially activated during osteogenic differentiation of BMSCs and ADSCs. (a–d) Expression of Wnt pathway-related genes and BMP pathway genes at different time points of BMSC (a and b) and ADSC (c and d) osteogenic differentiation was determined by real-time RT-PCR. (e) Smad1 and GSK3β mRNA expression in ADSCs and BMSCs during osteogenic differentiation was analyzed by real-time RT-PCR. (f and g) Western blot analysis of the markers of Wnt and BMP pathways during osteogenic differentiation of BMSCs (f) and ADSCs (g). β-Actin was used as the internal control. Relative protein abundance of each blots was normalized to the gray value of β-actin. (h and i) Immunofluorescence assay of p-Smad1 and active β-catenin in BMSC and ADSC transplants after 4 weeks of immunocompromised mice transplantation. The fluorescence intensity was quantified by Image Pro software (i). Scale bar: 200 μm. Results are represented as mean±S.D. **P<0.01, ***P<0.001 (n=3)
Figure 6
Figure 6
Wnt and BMP signaling pathways play different role in BMSC and ADSC osteogenic differentiation. (a–c) BMSCs were cultured in osteogenic medium with Dorsomorphin or DKK1. Then, ALP and alizarin red staining were performed separately after 7 and 14 days of induction (a). Alizarin red staining was quantified by spectrophotometer (b). Expression of Runx2, Alp and Ocn was measured by real-time RT-PCR (c). (d–f) ALP staining (d), alizarin red staining (d and e) and real-time RT-PCR analysis (f) in ADSCs cultured in osteogenic medium containing Dorsomorphin or DKK1. (g–l) ALP staining, alizarin red staining and real-time RT-PCR were performed in BMSCs (g–i) and ADSCs (j–l) cultured in osteogenic medium containing recombinant BMP2 or Wnt3a. Results are represented as mean±S.D. *P<0.05, **P<0.01, ***P<0.001 (n=3)
Figure 7
Figure 7
MiR-26a potently regulates different signaling pathways in BMSC and ADSC osteogenic differentiation. (a and b) BMSCs and ADSCs were transfected with negative control (miR-cont), miR-26a precursors (pre-miR-26a) and inhibitors (anti-miR-26a) for 48 h before osteogenic induction. Western blot was performed to analyze the protein level of active β-catenin or pSmad1 in BMSCs (a) and ADSCs (b) after 14 days of induction in vitro. Relative protein abundance of each blots was normalized to the gray value of β-actin. (c–f) BMSCs and ADSCs transfected with negative control (miR-cont), miR-26a precursors (pre-miR-26a) and inhibitors (anti-miR-26a) were implanted into nude mice for 4 weeks. Expression of pSmad1 and active β-catenin in transplants of BMSCs (c and d) and ADSCs (e and f) was detected by immunofluorescence assay, and quantified by Image Pro software. Scale bar: 200 μm. Results represent means±S.D. *P<0.05, **P<0.01, ***P<0.001 (n=3)
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