MicroRNA-15b shuttled by bone marrow mesenchymal stem cell-derived extracellular vesicles binds to WWP1 and promotes osteogenic differentiation

Arthritis Res Ther. 2020 Nov 16;22(1):269. doi: 10.1186/s13075-020-02316-7.

Abstract

Background: Osteogenic differentiation is an essential process for bone regeneration involving bone marrow mesenchymal stem cells (BMSCs). BMSC-secreted extracellular vesicles (EVs) enriched with microRNAs (miRs) have vital roles to play in mediating osteogenic differentiation. Therefore, this study aimed to explore the effect of BMSC-derived EVs loaded with miR-15b on osteogenic differentiation.

Methods: Human BMSCs (hBMSCs) were cultured and treated with plasmids overexpressing or knocking down KLF2, WWP1, and miR-15b to define the role of derived EVs in osteogenic differentiation in vitro. The expression of osteogenic differentiation-related marker was measured by Western blot analysis. The interaction among miR-15b, WWP1, and ubiquitination of KLF2 was investigated by dual-luciferase reporter, immunoprecipitation, and GST pull-down assays. Moreover, EVs from hBMSCs transfected with miR-15b inhibitor (EV-miR-15b inhibitor) were injected into ovariectomized rats to verify the effect of miR-15b on bone loss in vivo.

Results: WWP1 was downregulated, and KLF2 was upregulated during osteogenic differentiation. After co-culture with EVs, miR-15b expression was elevated and WWP1 expression was reduced in hBMSCs. Upregulation of miR-15b or KLF2 or downregulation of WWP1 or NF-κB increased ALP activity and cell mineralization, as well as osteogenic differentiation-related marker expression in hBMSCs. Mechanistically, miR-15b targeted and inhibited WWP1, thus attenuating KLF2 degradation and inhibiting NF-κB activity. Co-culture of EVs increased the bone volume and trabecular number, but decreased bone loss in ovariectomized rats, which could be reversed after treatment with EV-miR-15b inhibitor.

Conclusion: Collectively, BMSC-derived EVs loaded with miR-15b promoted osteogenic differentiation by impairing WWP1-mediated KLF2 ubiquitination and inactivating the NF-κB signaling pathway.

Keywords: Bone marrow mesenchymal stem cell; Extracellular vesicles; KLF2; NF-κB signaling pathway; Osteogenic differentiation; WWP1; microRNA-15b.

Publication types

  • Research Support, Non-U.S. Gov't
  • Retracted Publication

MeSH terms

  • Animals
  • Bone Marrow Cells
  • Cell Differentiation
  • Cells, Cultured
  • Extracellular Vesicles*
  • Kruppel-Like Transcription Factors / genetics
  • Mesenchymal Stem Cells*
  • MicroRNAs* / genetics
  • Osteogenesis / genetics
  • Rats
  • Ubiquitin-Protein Ligases

Substances

  • Klf2 protein, rat
  • Kruppel-Like Transcription Factors
  • MicroRNAs
  • WWP1 protein, human
  • Ubiquitin-Protein Ligases