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.
ABSTRACT
Our study showed that Signal transducer and activator of transcription (STAT)1 and STAT3 phosphorylation was firstly upregulated in the early stage of osteogenic differentiation (OD), and quickly eliminated in hours. Following with phosphorylation of STAT1/3, its downstream feedback regulator Suppressor of cytokine signaling 1 (SOCS1) protein also underwent a quick elevation. Further activation and deactivation of STAT1/3, by administrated with Colivelin and Nifuroxazide in Bone mesenchymal stem cells (BMSCs), increased and decreased SOCS1 expression, inhibited and promoted OD of BMSCs, respectively, as evidenced by Alizarin staining, alkaline phosphatase (ALP) activity, and determination of Run-related transcription factor 2 (RUNX2), Osteocalcin (OCN), ALP, and Bone sialoprotein (BSP). In addition, administration of Colivelin and Nifuroxazide caused and blocked inflammation and apoptosis of BMSCs. To further elucidate the role of STAT1/3-SOCS1 regulatory loop on OD of BMSCs, we overexpressed or silenced SOCS1 in BMSCs during OD. WB data showed that overexpression of SOCS1 repressed STAT1/3 phosphorylation, and knockdown of SOCS1 increased the phosphorylated STAT1/3. Further mechanism study showed that OD of BMSCs was elevated or reduced by SOCS1 overexpression or knockdown, respectively. The findings presenting indicated that the STAT1/3-SOCS1 axis may be exploited as an innovative strategy to enhance osteogenesis in regenerative medicine.
