Abstract
BackgroundAtrophic non-union fractures show no radiological evidence of callus formation within 3 months of fracture. microRNA dysregulation may underlie the dysfunctional osteogenesis in atrophic non-union fractures. Here, we aimed to analyze miR-1323 expression in human atrophic non-union fractures and examine miR-1323’s underlying mechanism of action in human mesenchymal stromal cells.MethodsHuman atrophic non-union and standard healing fracture specimens were examined using H&E and Alcian Blue staining, immunohistochemistry, qRT-PCR, immunoblotting, and ALP activity assays. The effects of miR-1323 mimics or inhibition on BMP4, SMAD4, osteogenesis-related proteins, ALP activity, and bone mineralization were analyzed in human mesenchymal stromal cells. Luciferase reporter assays were utilized to assay miR-1323’s binding to the 3'UTRs of BMP4 and SMAD4. The effects of miR-1323, BMP4, and SMAD4 were analyzed by siRNA and overexpression vectors. A rat femur fracture model was established to analyze the in vivo effects of antagomiR-1323 treatment.ResultsmiR-1323 was upregulated in human atrophic non-union fractures. Atrophic non-union was associated with downregulation of BMP4 and SMAD4 as well as the osteogenic markers ALP, collagen I, and RUNX2. In vitro, miR-1323 suppressed BMP4 and SMAD4 expression by binding to the 3'UTRs of BMP4 and SMAD4. Moreover, miR-1323’s inhibition of BMP4 and SMAD4 inhibited mesenchymal stromal cell osteogenic differentiation via modulating the nuclear translocation of the transcriptional co-activator TAZ. In vivo, antagomiR-1323 therapy facilitated the healing of fractures in a rat model of femoral fracture.ConclusionsThis evidence supports the miR-1323/BMP4 and miR-1323/SMAD4 axes as novel therapeutic targets for atrophic non-union fractures.
Highlights
Atrophic non-union is a fracture healing failure showing no radiological evidence of callus formation within 3 months of fracture [1]
We found that miR-1323 downregulates osteogenic differentiation of human mesenchymal stromal cells via directly targeting Bone morphogenic protein 4 (BMP4) and SMAD4
Results miR-1323 upregulation coupled with downregulation of BMP4/SMAD4 signaling and osteogenesis markers in atrophic non-union fractures The clinicodemographic characteristics of the human tissue donors are detailed in Additional Table 3
Summary
Atrophic non-union is a fracture healing failure showing no radiological evidence of callus formation within 3 months of fracture [1]. Fracture non-union has been associated with a decrease in bone mesenchymal stromal cell counts and a downregulation in their proliferative potential [3]. This is consistent with mesenchymal stromal cells’ potential for differentiation into osteoblasts [4] and their promotion of fracture healing [5]. Even though the significance of mesenchymal stromal cells in osteogenic differentiation has been established, their role in atrophic non-union is still unclear. Atrophic non-union fractures show no radiological evidence of callus formation within 3 months of fracture. We aimed to analyze miR-1323 expression in human atrophic non-union fractures and examine miR-1323’s underlying mechanism of action in human mesenchymal stromal cells
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