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MiR-1323 suppresses bone mesenchymal stromal cell osteogenesis and fracture healing via inhibiting BMP4/SMAD4 signaling.

ABSTRACT: BACKGROUND:Atrophic 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. METHODS:Human 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. RESULTS:miR-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. CONCLUSIONS:This evidence supports the miR-1323/BMP4 and miR-1323/SMAD4 axes as novel therapeutic targets for atrophic non-union fractures.

SUBMITTER: Xie H

PROVIDER: S-EPMC7322887 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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miR-1323 suppresses bone mesenchymal stromal cell osteogenesis and fracture healing via inhibiting BMP4/SMAD4 signaling.

Xie Hui H Liu Ming M Jin Yaofeng Y Lin Haiqing H Zhang Yushan Y Zheng Song S

Journal of orthopaedic surgery and research 20200629 1

<h4>Background</h4>Atrophic 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.<h4>Methods</h4>Human atrophic non-union and standard healing fracture specimens were examined using ...[more]

PMID: 32600409

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Project description:BackgroundMesenchymal stem cells (MSCs) are promising targets for therapeutic use in regenerative medicine and tissue engineering. In the previous study, we have found that MSCs could be reverted to a primitive stem cell population after in vitro induction of osteogenic and de-osteogenic differentiation (de-osteogenic differentiated MSCs, De-Os-MSCs). De-Os-MSCs showed improved cell survival and osteogenic potential. However, the underlying mechanism and its potential effect on fracture healing has not been explored.MethodsMSCs were isolated from the rat bone marrow. MicroRNAs were cloned into lentiviral vectors and transduced into MSCs to observe the effects on osteogenesis. The expression levels of marker genes were evaluated by quantitative RT-PCR. Ectopic bone formation model was used to evaluate the bone regeneration ability of mir-92b transduced MSCs in vivo. An open femur fracture model was established, and MSCs or De-Os-MSCs were administrated to the fracture sites. Histological, biomechanical and microCT analysis were used to evaluate the quality of bone.ResultsIn the present study, we found that mir-92b was significantly increased in the secretions of De-Os-MSCs. And mir-92b could promote the osteogenic differentiation potential of MSCs by activating pERK and JNK signaling pathways. The ectopic bone formation assay showed that MSCs overexpressing mir-92b formed more bone like tissues in vivo. Most importantly, we found local administration of De-Os-MSCs could accelerate fracture healing using an open femur fracture model in rats. The quality of bone property was much better as shown by microCT and biomechanical testing.ConclusionTaken together, our study demonstrated that mir-92b promoted osteogenesis of MSCs, which was partially accounted for the enhanced osteogenic differentiation potential of De-Os-MSCs. And De-Os-MSCs had shown better regenerative capacity in accelerating fracture healing when they were locally given.The translational potential of this articleDe-Os-MSCs could be used to accelerate fracture healing, and reduce the occurrence of delayed unions and non-unions.

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MiR-1323 suppresses bone mesenchymal stromal cell osteogenesis and fracture healing via inhibiting BMP4/SMAD4 signaling.

Publications

miR-1323 suppresses bone mesenchymal stromal cell osteogenesis and fracture healing via inhibiting BMP4/SMAD4 signaling.

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