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Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats
Published on: August 25, 2020
MicroRNA-182 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells by targeting Smad1
Abstract:
The present study aimed to screen abnormally expressed microRNAs (miRs/miRNAs) in patients with postmenopausal osteoporosis (POP) and explore their mechanisms via functional verification. Bone marrow mesenchymal stem cells (BMSCs) were extracted from healthy controls and patients with POP. Differences in osteogenic differentiation and proliferation of human BMSCs were compared between the two groups using Cell Counting Kit-8 (CCK-8) assay and alizarin red staining. A rat model of POP was established. Compared with patients with POP, human BMSCs in healthy controls had significantly enhanced viability at 24, 36, 48 and 72 h. The results of alizarin red staining revealed that the deposition of calcium minerals in human BMSCs were significantly lower in patients with POP. Based on miRNA microarray and reverse transcription-quantitative polymerase chain reaction (PCR) results, the expression levels of miR-7010 and miR-467c decreased, while miR-132 and miR-182 expression increased in the human BMSCs of patients with POP. Alizarin red staining showed that miR-182 markedly suppressed the osteogenic differentiation of primary rat BMSCs in rats. Western blotting and immunofluorescence assay revealed that miR-182 inhibited the expression of osteogenesis markers runt-related transcription factor 2, osterix and actinin-associated LIM protein. The results of the luciferase reporter assay showed that Smad1 is the direct target of miR-182. In rat primary BMSCs, Smad1 overexpression abolished the inhibitory effect of miR-182 on osteogenesis, indicating that miR-182 inhibits osteogenic differentiation of primary rat BMSCs in rats by targeting Smad1. Finally, in vivo experimental results showed that the biomechanical characteristics of bone tissues in POP rats were significantly enhanced by miR-182 inhibition, while they were significantly weakened by miR-182 overexpression. MiR-182 inhibits osteogenic differentiation of rat BMSCs, thus aggravating POP in rats.
Insights
MicroRNA-182 (miR-182) is upregulated in postmenopausal osteoporosis (POP) and inhibits bone formation by targeting Smad1. Inhibiting miR-182 improves bone characteristics in POP rat models.
Area of Science:
- Biomedical research
- Molecular biology
- Cell biology
Background:
- Postmenopausal osteoporosis (POP) is characterized by decreased bone mineral density and increased fracture risk.
- MicroRNAs (miRNAs) play crucial roles in bone metabolism, but their specific involvement in POP requires further elucidation.
Purpose of the Study:
- To identify differentially expressed miRNAs in patients with POP.
- To investigate the functional role and mechanism of miR-182 in osteogenic differentiation and POP progression.
Main Methods:
- Comparison of human bone marrow mesenchymal stem cells (BMSCs) from healthy controls and POP patients.
- In vitro assays including CCK-8, alizarin red staining, Western blotting, immunofluorescence, and luciferase reporter assay.
- Establishment of a rat model of POP for in vivo validation.
Main Results:
- BMSCs from POP patients exhibited reduced viability and osteogenic differentiation.
- miR-182 and miR-132 were upregulated, while miR-7010 and miR-467c were downregulated in POP BMSCs.
- miR-182 directly targeted Smad1, inhibiting osteogenic differentiation markers and bone formation.
- In vivo studies showed miR-182 inhibition improved bone biomechanical properties in POP rats.
Conclusions:
- miR-182 is a key regulator that inhibits osteogenic differentiation of BMSCs by targeting Smad1.
- Upregulation of miR-182 contributes to the pathogenesis of POP.
- Targeting miR-182 presents a potential therapeutic strategy for managing POP.
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