Knockdown of SIRT7 enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells partly via activation of the Wnt/β-catenin signaling pathway

Abstract

Sirtuin 7 (SIRT7) is a NAD⁺-dependent deacetylase in the sirtuin family. In a previous study, human bone marrow mesenchymal stem cells (hBMSCs) with reduced SIRT7 activity were developed to evaluate the effect of SIRT7 on osteogenesis. SIRT7 knockdown significantly enhanced osteoblast-specific gene expression, alkaline phosphatase activity, and mineral deposition in vitro. Additionally, SIRT7 knockdown upregulated β-catenin. The enhanced osteogenesis due to SIRT7 knockdown was partially rescued by a Wnt/β-catenin inhibitor. Furthermore, SIRT7 knockdown hBMSCs combined with a chitosan scaffold significantly promoted bone formation in a rat tibial defect model, as determined by imaging and histological examinations. These findings suggest that SIRT7 has an essential role in osteogenic differentiation of hBMSCs, partly by activation of the Wnt/β-catenin signaling pathway.

Figure 1

Endogenous SIRT7 expression and the construction of SIRT7-konckdown hBMSCs and lenti-control hBMSCs. (a–c) The endogenous expression of SIRT7 mRNA and protein were determined, respectively, by qPCR and western blotting analysis at days 0, 3, and 7 of osteogenic differentiation. (d) hBMSCs after lentiviral transfection and puromycin screening were observed under a normal microscope and a fluorescence microscope. (e–g) The mRNA and protein levels of SIRT7 were determined, respectively, by qPCR and western blotting analysis among the lenti-SIRT7, lenti-control group, and mock treated group. (h) The proliferation rate of hBMSCs was not significantly affected by SIRT7 knockdown. The mRNA and protein expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All the data were confirmed by three repeated tests. The data are expressed as means±S.D., *P<0.05 versus the lenti-control group

Figure 2

The knockdown of SIRT7 promoted osteogenic differentiation of hBMSCs. (a) The expression of RUNX2 mRNA was determined by qPCR at days 3 and 7 of osteogenic differentiation. (b) The expression of OSX mRNA. (c) The expression of OPN mRNA. (d) The expression of COL1A1 mRNA. (e–g) The expression of RUNX2 and COL1A1 proteins were determined by western blotting analysis after osteogenic differentiation for 3 and 7 days. (h) ALP in hMSCs was stained after the osteogenic differentiation for 3 and 7 days. (i) The ALP activity of hMSCs. (j) ARS after the osteogenic differentiation for 9 and 12 days. (k) Mineralization was quantified by the extraction of ARS-stained cells. All the data were confirmed by three repeated tests. Data were mean±S.D. *P<0.05 versus the lenti-control group. Scale bar=200 μm

Figure 3

Immunofluorescence staining showed the protein levels of RUNX2, SIRT7, and β-catenin. (a) The level of RUNX2 protein (red) at day 3 of osteogenic differentiation. (b) The level of SIRT7 protein (red) at day 3 of osteogenic differentiation. (c) The level of β-catenin protein (red) at day 3 of osteogenic differentiation. The nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole; blue). All the data were confirmed by three repeated tests. Scale bar=100 μm

Figure 4

The knockdown of SIRT7 upregulated the Wnt/β-catenin signaling pathway during osteogenesis. (a) The expression of β-catenin mRNA was determined by qPCR at days 3 and 7 of osteogenic differentiation. (b) The expression of GSK3β mRNA. (c) The expression of Axin mRNA. (d–f) The expression of active β-catenin and total β-catenin proteins were determined by western blotting analysis at days 3 and 7 of osteogenic differentiation. All the data were confirmed by three repeated tests. Data were mean±S.D. *P<0.05 versus the lenti-control group

Figure 5

The increased osteogenesis caused by SIRT7 konckdown could be rescued partially by the addition of a Wnt/β-catenin signaling inhibitor (DKK1). (a) The expression of β-catenin, GSK3β, and Axin mRNA in the lenti-control, lenti-control+DKK1, lenti-HSPA1A, and lenti-HSPA1A+DKK1 groups were determined by qPCR at days 3 of osteogenesis. (b) The expression of RUNX2, OSX, OPN, and COL1A1 mRNA at days 3 of osteogenesis. (c) The expression of RUNX2, COL1A1, active β-catenin, and total β-catenin proteins were determined by western blotting analysis at day 3 of osteogenesis. (d and e) ALP staining and activity at day 7 of osteogenesis. (f and g) ARS and quantitation at day 9 of osteogenesis. All the data were confirmed by three repeated tests. Data were mean±S.D. *P<0.05 versus the lenti-control group. #P<0.05 versus the lenti-SIRT7 group. Scale bar=200 μm

Figure 6

Radiographic and micro-CT analyses of the defect area at 6 weeks after surgery in each group. (a) Radiographic analysis. (b and c) Micro-CT images. (d–f) Micro-CT analyses of BV/TV, Tb.N, and Conn.D. All the data were confirmed by three repeated tests. Data were mean±S.D. *P<0.05 versus the blank group. Scale bar=1 mm

Figure 7

Histological evaluation of the defect area at 6 weeks after surgery in each group. (a–d) HE staining. Scale bar=500 μm. (e–h) Safranin O and fast green staining. (i–l) Masson staining. All the data were confirmed by three repeated tests. Scale bar=500 μm