Exosomal miR-130a-3p regulates osteogenic differentiation of Human Adipose-Derived stem cells through mediating SIRT7/Wnt/β-catenin axis

Abstract

Objectives: It is of profound significance for clinical bone regeneration to clarify the specific molecular mechanism from which we found that osteogenic differentiation of adipose-derived stem cells (ADSCs) will be probably promoted by exosomes.

Materials and methods: By means of lentiviral transfection, miR-130a-3p overexpression and knockdown ADSCs were constructed. Alizarin Red S was used to detect the calcium deposits, and qPCR was used to detect osteogenesis-related genes, to verify the effect of miR-130a-3p on the osteogenic differentiation of ADSCs. CCK-8 was used to detect the effect of miR-130a-3p on the proliferation of ADSCs. The target binding between miR-130a-3p and SIRT7 was verified by dual-luciferase reporter gene assay. Furthermore, the role of Wnt signalling pathway in the regulation of ADSCs osteogenesis and differentiation by miR-130a-3p was further verified by detecting osteogenic-related genes and proteins and alkaline phosphatase activity.

Results: (a) Overexpression of miR-130a-3p can enhance the osteogenic differentiation of ADSCs while reducing protein and mRNA levels of SIRT7, a target of miR-130a-3p. (b) Our study further found that overexpression of miR-130a-3p leads to down-regulation of SIRT7 expression with up-regulation of Wnt signalling pathway-associated protein. (c) Overexpression of miR-130a-3p inhibited proliferation of ADSCs, while knockdown promoted it.

Conclusions: The obtained findings indicate that exosomal miR-130a-3p can promote osteogenic differentiation of ADSCs partly by mediating SIRT7/Wnt/β-catenin axis, which will hence promote the application of exosomal microRNA in the field of bone regeneration.

Keywords: Adipose-derived stem cells; Wnt/β-catenin; bone regeneration; exosomes; miR-130a-3p.

Figure 1

Characteristics of ADSCs. A, The expression of special surface markers detected by flow cytometry analysis. B, The ability of ADSCs to differentiate into osteoblasts confirmed by ARS. C, The ability of ADSCs to differentiate into adipocytes identified by oil red O staining. D, The ability of ADSCs to differentiate into chondrocytes identified by alcian blue. Notes: ARS:Alizarin Red S

Figure 2

Identification of exosomes derived from ADSCs and the expression of miR‐130a‐3p in ADSCs‐derived exosomes. A, The size and morphology of exosomes observed using TEM. Scale bar: 100 nm. B, Specific markers of exosomes detected by Western blot. C, Nanoparticle tracking analysis of exosomes. D, qPCR analyses expression of miR‐130a‐3p in Exos_D0 and Exos_D14. *represents significant differences between Exos_D0 and Exos_D14. *P < .05

Figure 3

Construction of miR‐130a‐3p overexpressing ADSCs, miR‐130a‐3p knockdown ADSCs and lenti‐control ADSCs. A, 24 h after transfection, ADSCs observed under a normal microscope and an inverted fluorescence microscope (magnification, ×4). B, The expression of miR‐130a‐3p quantified by qPCR. Notes: *represents significant differences between the control group and other groups; #represents significant differences between lenti‐control and overexpression groups, or lenti‐control and knockdown groups. *P < .05; #P < .05

Figure 4

The effect of miR‐130a‐3p on proliferation and differentiation of ADSCs. A, The proliferation rate detected by CCK8. B‐D, The expression of ALP, RUNX2 and Osterix mRNA analysed by qPCR on the third day of induction. E, Calcium deposits detected by ARS assay. Scale bar: 100 μm. Notes: *represents significant differences between control and other groups; #represents significant differences between lenti‐control and overexpression groups, or lenti‐control and knockdown groups. *P < .05; #P < .05

Figure 5

Target binding between miR‐130a‐3p and SIRT7. A, MiR‐130a‐3p predicted to bind to 3’UTR of SIRT7 by bioinformatics analysis. B, The expression of SIRT7 protein analysed by Western blot. C, Relative intensity analyses of Western blot results of SIRT7. D, qPCR performed to detect SIRT7 mRNA. E, 3’UTR of wild‐type SIRT7 mRNA and 3ʹUTR of mutant‐type SIRT7 mRNA cloned into luciferase vectors. F, Luciferase activity was detected. Notes: C: blank control; PC: miR‐130a‐3p positive control; WT:SIRT7 wild type; MUT:SIRT7 mutant type; NC: negative control. *represents significant differences between control and other groups; #represents significant differences between lenti‐control and overexpression groups, or lenti‐control and knockdown groups; and *represents significant differences between NC and hsa‐miR‐130a‐3p groups. *P < .05, #P < .05, *P < .05

Figure 6

The mechanism of miR‐130a‐3p promoting osteogenic differentiation of ADSCs through Wnt signalling pathway. A‐B, The expression of osteogenic‐related protein and key protein of Wnt signalling pathway analysed by Western blot on 3rd and 7th day. C‐D, The expression of ALP and RUNX2 mRNA detected by qPCR. E‐F, ALP activity detected by ALP assay kit. G, The diagram of the mechanism that exosomal miR‐130a‐3p regulates osteogenic differentiation of ADSCs by mediating Wnt signalling pathway. Notes:*represents significant differences between control and other groups without DKK1 treatment; *represents significant differences between no treatment and DKK1 treatment group; and #represents significant differences between lenti‐control and overexpression groups, or lenti‐control and knockdown groups without DKK1 treatment. *P < .05; *P < .05; #P < .05