miR-140-3p regulates the osteogenic differentiation ability of bone marrow mesenchymal stem cells by targeting spred2-mediated autophagy

Abstract

Understanding the function and regulatory mechanism of miR-140-3p on the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Alizarin Red staining, Alkaline phosphatase (ALP) staining, and ALP activity were used to detect the ability osteogenic differentiation. miR-140-3p or Spred2 overexpression into BMSCs using lentiviral vectors and the result were analyzed by Reverse transcription quantitative polymerase chain reaction (RT-qPCR). The relation between miR-140-3p and Spred2 was examined by luciferase reporter assay. CCK8 assay was used to detect the proliferation of BMSCs. RT-qPCR and Western blot analysis were both used to detect altered gene and protein in osteogenic differentiation of BMSCs, respectively. The BMSCs which were induced for 21 days were analyzed by Alizarin Red staining, (ALP) staining and ALP activity. RT-qPCR analysis showed that overexpressed miR-140-3p promotes osteogenic differentiation. Western blots results indicated that the overexpression of Spred2 suppressed miR-140-3p. Luciferase reporter assay indicated that Spred2 can integrate with miR-140-3p directly. Meanwhile, the protein level of ALP, OCN, and Runx2, the markers of chondrogenesis, was increased when miR-140-3p increased or Spred2 overexpressed in the osteoinductive medium applied to the BMSCs. Our study demonstrated the association between miR-140-3p and Spred2 in osteogenic differentiation of BMSCs for the first time. Furthermore, our detections also revealed that Spred2-induced autophagic signaling accelerates the progress of osteogenic differentiation ability of BMSCs.

Keywords: Autophagy; Bone mesenchymal stem cells (BMSCs); MiR-140-3p; Osteogenic differentiation.