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Updated: Jun 19, 2026
Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice
Published on: January 6, 2018
A novel miR-466l-3p/FGF23 axis promotes osteogenic differentiation of human bone marrow mesenchymal stem cells
1Key Laboratory of Endocrine Glucose & Lipids Metabolism and Brain Aging, Ministry of Education, Department of Endocrinology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250021, China; Shandong Key Laboratory of Endocrinology and Lipid Metabolism, Jinan, Shandong 250021, China; Shandong Institute of Endocrine and Metabolic Diseases, Jinan, Shandong 250021, China.
Background:
MicroRNAs (miRNAs) regulate osteogenic differentiation processes and influence the development of osteoporosis (OP). This study aimed to investigate the potential role of miR-466 l-3p in OP.
Methods:
The expression levels of miR-466 l-3p and fibroblast growth factor 23 (FGF23) were quantified in the trabeculae of the femoral neck of 40 individuals with or without OP using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The impact of miR-466 l-3p or FGF23 expression on cell proliferation and the expression levels of runt-related transcription factor 2 (RUNX2), type I collagen (Col1), osteocalcin (OCN), osterix (OSX) and dentin matrix protein 1 (DMP1) was quantified in human bone marrow mesenchymal stem cells (hBMSCs) overexpressing miR-466 l-3p. Furthermore, alkaline phosphatase (ALP) staining and alizarin red staining were performed to measure ALP activity and the levels of calcium deposition, respectively. In addition, bioinformatics analysis, luciferase reporter assays, and RNA pull-down assays were conducted to explore the molecular mechanisms underlying the effects of miR-466 l-3p and FGF23 in osteogenic differentiation of hBMSCs.
Results:
The expression levels of miR-466 l-3p were significantly lower in femoral neck trabeculae of patients with OP than in the control cohort, whereas FGF23 levels exhibited the opposite trend. Furthermore, miR-466 l-3p levels were upregulated and FGF23 levels were downregulated in hBMSCs during osteogenic differentiation. Moreover, the high miR-466 l-3p expression enhanced the mRNA expression of RUNX2, Col1, OCN, OSX and DMP1, as well as cell proliferation, ALP activity, and calcium deposition in hBMSCs. FGF23 was found to be a direct target of miR-466 l-3p. FGF23 overexpression downregulated the expression of osteoblast markers and inhibited the osteogenic differentiation induced by miR-466 l-3p overexpression. qRT-PCR and Western blot assays showed that miR-466 l-3p overexpression decreased the expression levels of mRNAs and proteins associated with the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, whereas FGF23 upregulation exhibited the opposite trend.
Conclusion:
In conclusion, these findings suggest that miR-466 l-3p enhances the osteogenic differentiation of hBMSCs by suppressing FGF23 expression, ultimately preventing OP.
Insights
MicroRNA miR-466l-3p enhances bone formation by downregulating FGF23, offering a potential therapeutic target for osteoporosis (OP). This study reveals miR-466l-3p's crucial role in promoting osteogenic differentiation and preventing OP.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- MicroRNAs (miRNAs) are key regulators of osteogenic differentiation and are implicated in osteoporosis (OP) development.
- The specific role of miR-466l-3p in OP pathogenesis remains largely unexplored.
Purpose of the Study:
- To investigate the function of miR-466l-3p in the context of osteoporosis.
- To elucidate the molecular mechanisms by which miR-466l-3p influences osteogenic differentiation.
Main Methods:
- Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to measure miR-466l-3p and FGF23 expression in patient samples and cell cultures.
- Overexpression studies in human bone marrow mesenchymal stem cells (hBMSCs) to assess the impact of miR-466l-3p on osteogenic markers and cell proliferation.
- Bioinformatics analysis, luciferase reporter assays, and RNA pull-down assays to determine direct interactions between miR-466l-3p and FGF23.
- Western blot analysis to examine signaling pathway involvement (PI3K/AKT/mTOR).
Main Results:
- miR-466l-3p expression was decreased, while FGF23 expression was increased in osteoporotic patients' femoral neck trabeculae.
- miR-466l-3p overexpression in hBMSCs promoted osteogenic differentiation markers (RUNX2, Col1, OCN, OSX, DMP1), cell proliferation, ALP activity, and calcium deposition.
- FGF23 was identified as a direct target of miR-466l-3p; FGF23 overexpression counteracted the osteogenic effects of miR-466l-3p.
- miR-466l-3p suppressed the PI3K/AKT/mTOR signaling pathway, whereas FGF23 upregulated it.
Conclusions:
- miR-466l-3p promotes osteogenic differentiation in hBMSCs by directly inhibiting FGF23 expression.
- This regulatory axis suggests that miR-466l-3p plays a protective role against osteoporosis by enhancing bone formation.
