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miR-129-5p Promotes Osteogenic Differentiation of BMSCs and Bone Regeneration via Repressing Dkk3
Changming Zhao1,2, Yulin Gu1,3, Yan Wang4
1Medical School of Chinese PLA, Beijing 100853, China.
Objective:
Accumulating evidence indicates that microRNAs (miRNAs) play crucial roles in osteogenic differentiation. However, the associated mechanisms remain elusive. This paper is aimed at exploring the role of miR-129-5p in regulating bone marrow mesenchymal stem cell (BMSC) differentiation and bone regeneration in vivo and in vitro.
Methods:
BMSCs were transduced by miR-129-5p mimic, miR-129-5p inhibitor, and negative control lentivirus. The ability of BMSC differentiation to osteoblast was tested by alkaline phosphatase (ALP) and alizarin red staining (ARS). The expression of osteogenic genes (Runx2, Bmp2, and OCN) was examined via quantitative RT-PCR and western blot. A mouse model of calvaria defect was investigated by Micro-CT, immunohistochemistry, and histological examination. The luciferase reporter gene assay was performed to confirm the binding between Dkk3 and miR-129-5p. For the transfection experiments, lipofectamine 3000 was used to transfect pcDNA-Dkk3 into BMSCs to overexpress Dkk3. Coimmunoprecipitation and immunofluorescent localization assay were included for exploring the role of Dkk3 and β-catenin.
Results:
miR-129-5p was induced in BMSCs and MSC cell line C3H10T1/2 cells under osteogenic medium. Overexpression of miR-129-5p significantly promoted osteogenic differentiation of BMSCs in vitro. Moreover, BMSCs transduced with miR-129-5p mimic exhibited better bone regeneration compared with BMSCs transduced with control counterpart in vivo. Luciferase and western blot data showed that Dickkopf3 (Dkk3) is a target gene of miR-129-5p and the expression of Dkk3 was inhibited in BMSCs transduced with miR-129-5p mimic but enhanced in BMSCs transduced with miR-129-5p inhibitor. In addition, Dkk3 interacted with β-catenin directly.
Conclusions:
miR-129-5p promotes osteogenic differentiation of BMSCs and bone regeneration, and miR-129-5p/Dkk3 axis may be new potential targets for the treatment of bone defect and bone loss.
Insights
MicroRNA-129-5p enhances bone marrow mesenchymal stem cell differentiation and bone regeneration. Targeting the miR-129-5p/Dkk3 pathway shows promise for treating bone defects and bone loss.
Area of Science:
- Stem Cell Biology
- Molecular Biology
- Regenerative Medicine
Background:
- MicroRNAs (miRNAs) are critical regulators of osteogenic differentiation.
- The precise mechanisms underlying miRNA involvement in bone formation are not fully understood.
Purpose of the Study:
- To investigate the role of miR-129-5p in regulating bone marrow mesenchymal stem cell (BMSC) differentiation.
- To explore the impact of miR-129-5p on bone regeneration in vivo and in vitro.
Main Methods:
- Lentiviral transduction of BMSCs with miR-129-5p mimic or inhibitor.
- Assessment of osteogenic differentiation using alkaline phosphatase and alizarin red staining.
- Quantitative RT-PCR and western blot for osteogenic gene expression.
- In vivo mouse calvarial defect model with Micro-CT and histological analysis.
- Luciferase reporter assays to identify Dkk3 as a direct target of miR-129-5p.
Main Results:
- miR-129-5p expression increased under osteogenic conditions.
- Overexpression of miR-129-5p significantly promoted BMSC osteogenic differentiation in vitro.
- BMSCs with miR-129-5p mimic showed enhanced bone regeneration in vivo.
- miR-129-5p directly targets Dickkopf3 (Dkk3), inhibiting its expression.
- Dkk3 was found to interact with β-catenin.
Conclusions:
- miR-129-5p promotes BMSC osteogenic differentiation and bone regeneration.
- The miR-129-5p/Dkk3 axis represents a potential therapeutic target for bone defects and bone loss.
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