MiR-143-3p regulates early cartilage differentiation of BMSCs and promotes cartilage damage repair through targeting BMPR2

OBJECTIVE: The aim of this study was to explore the role of microRNA-143-3p (miR-143-3p) in cartilage injury, and to investigate the possible underlying mechanism.

MATERIALS AND METHODS: A chondrogenic differentiation cell model was established in bone marrow mesenchymal stem cells (BMSCs). The mRNA expression levels of runt-related transcription factor 2 (RUNX2), miR-143-3p and bone morphogenetic protein 2 (BMPR2) in BMSCs were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) after 0 d, 5 d and 10 d, respectively. Mesenchymal stem cells (MSCs) were transfected with miR-143-3p mimics and its control in accordance with the liposome method. Alcian blue colorimetric assay was used to evaluate proteoglycan deposition of MSCs. Meanwhile, qRT-PCR and Western blot were performed to analyze the expression levels of ACAN and COL2A1. Luciferase reporter gene assay was applied to verify the binding status of miR-143-3p and BMPR2 3'UTR. Also, proteoglycan deposition and the expression of ACAN and COL2A1 were detected after simultaneous transfection of miR-143-3p mimics and BMPR2 overexpression plasmid.

RESULTS: 0 d, 5 d and 10 d after inducing cartilage differentiation, the mRNA expression levels of RUNX2 and BMPR2 were markedly increased. However, the expression level of miR-143-3p was significantly decreased with the prolongation of induction period. After transfection with miR-143-3p mimics, the level of miR-143-3p in MSCs was remarkably increased. Alcian blue colorimetric assay and staining assay showed that the deposition of proteoglycans in the mimics group was significantly lower than that of the control group. Meanwhile, after overexpressing miR-143-3p, the levels of cartilage differentiation marker proteins including ACAN and COL2A1 were remarkably reduced. Luciferase report gene assay indicated that miR-143-3p could negatively regulate BMPR2 by binding to its 3'UTR. In addition, overexpression of BMPR2 could strikingly reverse the above effects of overexpressed miR-143-3p.

CONCLUSIONS: During chondrogenic differentiation, the level of miR-143-3p was decreased. Moreover, miR-143-3p could regulate the differentiation process by targeting BMPR2 in BMSCs.

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