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.
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.
Authors: Chiara Mazziotta; Carmen Lanzillotti; Maria Rosa Iaquinta; Francesca Taraballi; Elena Torreggiani; John Charles Rotondo; Lucia Otòn-Gonzalez; Elisa Mazzoni; Francesca Frontini; Ilaria Bononi; Monica De Mattei; Mauro Tognon; Fernanda Martini Journal: Int J Mol Sci Date: 2021-02-27 Impact factor: 6.208