Bone Marrow Mesenchymal Stem Cell-Derived Exosomal miR-25 Regulates the Ubiquitination and Degradation of Runx2 by SMURF1 to Promote Fracture Healing in Mice

Abstract

Recent evidence has demonstrated that mesenchymal stem cells (MSCs) can release a large number of functionally specific microRNA (miRNA) microvesicles that play a role in promoting osteogenic differentiation, but the specific mechanism is not yet clear. Under such context, this study aims to elucidate the mechanism of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exo) promoting fracture healing in mice. We isolated and identified the BMSC-Exo. Bioinformatics analysis predicted high expression of miRNA in exosomes and verified the transfer of miR-25 in exosomes by immunofluorescence. Targeting relationship between miR-25 and Smad ubiquitination regulatory factor-1 (SMURF1) was predicted and verified by dual-luciferase reporter gene assay. Immunoprecipitation and protein stability assays were used to detect Runt-related transcription factor 2 (Runx2) ubiquitination and the effect of SMURF1 on Runx2 ubiquitination, respectively. The effect of miR-25 in BMSC-Exo on fracture healing in mice was assessed using X-ray imaging. alkaline phosphatase, alizarin red staining, EdU, CCK-8, and Transwell were used to evaluate the effects of exosomes transferred miR-25 on osteogenic differentiation, proliferation, and migration of osteoblasts. Bioinformatics analysis predicted that miR-25 expression in exosomes increased significantly. Moreover, the targeted regulation of SMURF1 by miR-25 was verified. SMURF1 inhibited Runx2 protein expression by promoting ubiquitination degradation of Runx2. Notably, miR-25 secreted by BMSC-Exo can accelerate osteogenic differentiation, proliferation, and migration of osteoblasts through SMURF1/Runx2 axis. Our results demonstrate that miR-25 in BMSC-Exo regulates the ubiquitination degradation of Runx2 by SMURF1 to promote fracture healing in mice.

The protective effects of pericyte-derived microvesicles on vascular endothelial functions via CTGF delivery in sepsis

“…On the other hand, MVs are natural membrane vesicles secreted, thus they are proved to have lower immunogenicity and toxicity, and are more efficiently absorbed by target cells than other synthetic delivery vehicles, such as liposomes, micelles, dendrimers and nanocapsules [ 22 ]. It has been proved that MMVs may promote the proliferation of osteoblasts through regulating transcription activity and expression level of genes [ 23 , 24 ]. There were studies to report pericytes-derived microvesicles (PMVs) from human cerebrovascular sources may carry neuroprotective factors, including VEGF, BDNF, and PLGF, to reduce nerve cell damage [ 25 – 27 ].…”

Educating EVs to Improve Bone Regeneration: Getting Closer to the Clinic

“…In recipient MSCs, miR-335 inhibited the in vitro expression of the pro-osteoclastogenic protein VAPB, promoting the activation of Wnt/β-catenin pathway, and therefore stimulating the osteogenesis of MSCs, plus, finally the in vivo fracture repair in mice models [35]. BM-MSCs-EVs also have been shown to promote fracture healing in mice mediated by enriched miR-25, which facilitates osteogenic differentiation, proliferation, and migration of osteoblasts by inhibiting RUNX2 degradation through downregulation of the ubiquitin ligase SMURF1 [36].…”

miRNAs Related to Different Processes of Fracture Healing: An Integrative Overview

“…Presumably by enhancing Rac1 expression, miR-25 activated PI3K/AKT and c-Jun N-terminal kinase pathways, leading to enhanced osteoblastic viability and migration, as well as increased expression levels of osteogenic marker genes RUNX2 and OCN (80). Research by Jiang et al showed that miR-25 also targeted the expression SMURF1, thereby enhancing RUNX2 signalling and subsequently promoting osteogenic differentiation (81). Hu et al examined the interaction of miR-26b with the Wnt-signalling pathway in rat BMSCs.…”