Abstract
BackgroundMicroRNAs (miRNAs) play a role in regulating osteogenic differentiation (OD) of mesenchymal stem cells by inhibiting mRNAs translation under cyclic strain. miR-503-3p was downregulated in OD of human adipose-derived stem cells (hASCs) in vivo under cyclic strain in our previous study, while it might target the Wnt/β-catenin (W-β) pathway. In this study, we explored miR-503-3p’s role in OD of hASCs under cyclic strain.MethodsOD of hASCs was induced by cyclic strain. Bioinformatic and dual luciferase analyses were used to confirm the relationship between Wnt2/Wnt7b and miR-503-3p. Immunofluorescence was used to detect the effect of miR-503-3p on Wnt2/Wnt7b and β-catenin in hASCs transfected with miR-503-3p mimic and inhibitor. Mimic, inhibitor, and small interfering RNA (siRNA) transfected in hASCs to against Wnt2 and Wnt7b. Quantitative real-time PCR (RT-PCR) and western blot were used to examine the OD and W-β pathway at the mRNA and protein levels, respectively. Immunofluorescence was performed to locate β-catenin. ALP activity and calcium were detected by colorimetric assay.ResultsResults of immunophenotypes by flow cytometry and multi-lineage potential confirmed that the cultured cells were hASCs. Results of luciferase reporter assay indicated that miR-503-3p could regulate the expression levels of Wnt2 and Wnt7b by targeting their respective 3′-untranslated region (UTR). Under cyclic strain, gain- or loss-function of miR-503-3p studies by mimic and inhibitor revealed that decreasing expression of miR-503-3p could significantly bring about promotion of OD of hASCs, whereas increased expression of miR-503-3p inhibited OD. Furthermore, miR-503-3p high-expression reduced the activity of the W-β pathway, as indicated by lowering expression of Wnt2 and Wnt7b, inactive β-catenin in miR-503-3p-treated hASCs. By contrast, miR-503-3p inhibition activated the W-β pathway.ConclusionsCollectively, our findings indicate that miR-503-3p is a negative factor in regulating W-β pathway by Wnt2 and Wnt7b, which inhibit the OD of hASCs under cyclic strain.
Highlights
In 2001, human adiposederived stem cells (hASCs) were extracted for the first time by digestion of human adipose tissue [1]. hASCs have extensive proliferative potential and the ability to differentiate toward adipogenic, osteogenic, chondrogenic, and myogenic lineages [2, 3]
The results showed that hASCs transfected with miR503-3p mimic and wt-Wnt2 significantly reduced the luciferase activity 1.63 ± 0.39-fold (F) (p = 0.024) when compared with hASCs transfected with miR-Ctrl mimic (MCM) and wt-Wnt2 (Fig. 2d)
The luciferase activity of hASCs transfected with miR-503-3p mimic and mut-Wnt2 had no statistical significance, compared with hASCs transfected with MCM and mu-Wnt2 (p = 0.377) (Fig. 2d), indicating the inhabitation of luciferase activity regulated by miR-503-3p mimic was broken by the mut-Wnt2
Summary
In 2001, hASCs were extracted for the first time by digestion of human adipose tissue [1]. hASCs have extensive proliferative potential and the ability to differentiate toward adipogenic, osteogenic, chondrogenic, and myogenic lineages [2, 3]. Current studies suggest that hASCs may be an important new source of seed cells in bone tissue engineering. An important aspect of bone regeneration that requires further study is to determine how in vitro OD of hASCs can be effectively promoted [6,7,8]. Studies indicate that tensile strain can effectively promote OD of hASCs in vitro [6, 9]; these results have been used to promote OD in bone regeneration [6,7,8]. MicroRNAs (miRNAs) play a role in regulating osteogenic differentiation (OD) of mesenchymal stem cells by inhibiting mRNAs translation under cyclic strain. MiR-503-3p was downregulated in OD of human adiposederived stem cells (hASCs) in vivo under cyclic strain in our previous study, while it might target the Wnt/β-catenin (W-β) pathway. We explored miR-503-3p’s role in OD of hASCs under cyclic strain
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