Regenerative Therapy (Jun 2022)
MiRNA-320a-5p contributes to the homeostasis of osteogenesis and adipogenesis in bone marrow mesenchymal stem cell
Abstract
Objective: A number of miRNAs and their targets were dragged in the differentiation of bone marrow mesenchymal stem cells (BMSCs). We aimed to elaborate the underlying molecular mechanisms of miRNA-320a in the osteoblast and adipocyte differentiation. Methods: Trauma-induced osteonecrosis of the femoral head (TIONFH) and normal control samples (n = 10 for each group) were collected, followed by miRNA chip analysis to identify the differentially expressed miRNAs. H&E staining was used to observe the pathological development of TIONFH. Lentiviral vector was used for overexpression and inhibition of miRNA-320a in vitro. Quantitative real-time PCR (qPCR), Western blotting and immunohistochemistry staining were employed to determine the expression of interested genes at mRNA or protein level. Luciferase report assay was employed to determine the binding of miRNA-320a and RUNX2. Alkaline phosphatase (ALP) and Alizarin red staining were performed to observe the osteogenesis and Oil red O staining were conducted to visualize the adipogenesis. Results: Expression of miRNA-320a was up-regulated while RUNX2 expression was down-regulated in TIONFH than Normal control. Luciferase report assay confirmed that miRNA-320a directly targeted to the 3′UTR of RUNX2. miRNA-320a overexpression significantly declined the expressions of osteogenesis-related markers: RUNX2, OSTERIX, Collagen I, Osteocalcin and Osteopontin. ALP and Alizarin red staining confirmed the inhibition function of miRNA-320a in osteogenesis of BMSCs. miRNA-320a inhibition significantly decreased the expression of adipogenesis-related markers: AP2, C/EBPα, FABP4 and PPARγ. Oil Red O staining confirmed the miRNA-320a inhibition reduced adipogenesis of BMSCs. Conclusions: miRNA-320a inhibits osteoblast differentiation via targeting RUNX2 and promotes adipocyte differentiation of BMSCs.
