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Published on: February 24, 2017
MiR-15b ameliorates SONFH by targeting Smad7 and inhibiting osteogenic differentiation of BMSCs
1Department of Orthopedics, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China. 13506989645@163.com.
Objective:
The aim of this study was to explore the role of micro ribonucleic acid (miR)-15b in steroid-induced osteonecrosis of the femoral head (SONFH) and its potential mechanism.
Patients And Methods:
Bone marrow tissues were collected from 5 patients with glucocorticoid (GC)-induced ONFH (GC-ONFH, GC group) and 5 patients with secondary ONFH (control group) undergoing total hip replacement in our hospital from July 2016 to August 2017. Subsequently, bone marrow mesenchymal stem cells (BMSCs) were separated from bone marrow extracted and cultured in vitro. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assay was used to detect differentially expressed miRNAs in BMSCs of patients in GC group and control group. BMSCs were treated with different concentrations of GC. Next, the effect of GC on tmiR-15b expression level was detected via qRT-PCR. Alizarin red staining assay was performed to evaluate the effect of miR-15b on osteogenic differentiation of BMSCs. Meanwhile, the potential targets of miR-15b were predicted using bioinformatics software and validated through luciferase reporter gene assay, respectively. Additionally, Western blotting was conducted to determine the effect of miR-15b on the protein expression of the transforming growth factor beta (TGF-β) signaling pathway.
Results:
Flow cytometry demonstrated that the proportion of cluster of differentiation 44 (CD44)-positive cells was 99.7%, while that of CD45-positive cells was only 0.17% in cultured BMSCs. This suggested that the purity of BMSCs was relatively high. QRT-PCR assay indicated that the expression level of miR-15b declined significantly in BMSCs of GC group when compared with control group (p<0.01). The osteogenic differentiation capacity of BMSCs was significantly strengthened in GC group compared with control group (p<0.01). Subsequent qRT-PCR assay revealed that GC down-regulated the expression level of miR-15b in a dose-dependent manner. Besides, the osteogenic differentiation capacity of cells was remarkably strengthened in miR-15b mimic treatment group when compared with control group (p<0.01). Bioinformatics software (TargetScan) predicted that drosophila mothers against decapentaplegic protein 7 (Smad7) might be a potential target of miR-15b, which was indicated by luciferase reporter gene assay. In comparison with control group, miR-15b mimic treatment group exhibited significantly down-regulated protein expression level of Smad7, increased expression level of phosphorylated (p)-Smad2/3 and up-regulated messenger RNA (mRNA) expression level of runt-related transcription factor 2 (Runx2). However, the protein expression level of Smad7 and p-Smad2/3 and the mRNA expression level of Runx2 exhibited opposite trends in miR-15b inhibitor treatment group.
Conclusions:
MiR-15b relieves SONFH by targeting Smad7 and repressing osteogenic differentiation of BMSCs.
Insights
Micro ribonucleic acid (miR)-15b plays a crucial role in steroid-induced osteonecrosis of the femoral head (SONFH). This study found that miR-15b represses osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by targeting Smad7, offering potential therapeutic insights.
Area of Science:
- Biomedical Science
- Molecular Biology
- Stem Cell Research
Background:
- Steroid-induced osteonecrosis of the femoral head (SONFH) is a debilitating condition.
- The precise molecular mechanisms underlying SONFH, particularly the role of microRNAs, remain incompletely understood.
Purpose of the Study:
- To investigate the role of micro ribonucleic acid (miR)-15b in steroid-induced osteonecrosis of the femoral head (SONFH).
- To elucidate the potential molecular mechanism by which miR-15b influences SONFH pathogenesis.
Main Methods:
- Collected bone marrow tissues from patients with glucocorticoid (GC)-induced ONFH and controls.
- Isolated and cultured bone marrow mesenchymal stem cells (BMSCs).
- Utilized quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), Alizarin red staining, bioinformatics, luciferase reporter gene assay, and Western blotting to analyze miR-15b expression, osteogenic differentiation, and target interactions.
Main Results:
- Glucocorticoid treatment significantly down-regulated miR-15b expression in BMSCs in a dose-dependent manner.
- miR-15b mimic treatment enhanced osteogenic differentiation, while miR-15b inhibitor treatment repressed it.
- Smad7 was identified as a direct target of miR-15b, and miR-15b modulated the transforming growth factor beta (TGF-β) signaling pathway components.
Conclusions:
- miR-15b plays a protective role in steroid-induced osteonecrosis of the femoral head.
- miR-15b represses osteogenic differentiation of BMSCs by targeting Smad7.
- Modulating miR-15b levels may represent a therapeutic strategy for SONFH.
