Abstract
AimTo investigate the dysregulation of microRNAs (miRNAs) during the differentiation of osteoclasts and the precise roles of miR-125a-5p in the differentiation of osteoclasts.MethodsThe cell model of RAW 264.7 osteoclast precursor cell differentiation induced by RANKL plus M-CSF stimulation was established. During the early stage of osteoclast differentiation, miRNA expression profiles were detected using the biochip technique and analyzed by cluster analysis. TargetScan, miRTarBase and miRDB database analysis was applied to find the key target genes of miR-125a-5p. A dual luciferase experiment was conducted to identify the direct target of miR-125a-5p. MiR-125a-5p mimic transfection and anti-miR-125-5p treatment were conducted to verify the role of miR-125q-5p in osteoclast differentiation. The levels of triiodothyronine receptor auxiliary protein (TRAP), matrix metallopeptidase 2 (MMP-2), MMP-9 and cathepsin K were analyzed by qRT-PCR and western blot assay. The expression levels of MMP-2 and MMP-9 were determined using western blotting and immunofluorescence assay. The migration and invasion of RAW 264.7 cells were assessed by wound healing and Transwell invasion assays. The proliferation of RAW 264.7 osteoclast precursor cells was detected using MTT assay.ResultsThere were 44 microRNAs differently expressed during the differentiation of RAW 264.7 osteoclast precursor cells into osteoclasts, 35 of which were up-regulated and 9 were down-regulated. By luciferase reporter assay, it was confirmed that the TNF receptor superfamily member 1B gene (TNFRSF1B) was the target gene of miR-125a-5p. Up-regulation of miR-125a-5p inhibited TNFRSF1B protein expression and promoted osteoclast differentiation whereas down-regulation of miR-125a-5p caused completely opposite results.ConclusionsIn conclusion, overexpression of miR-125a-5p suppresses the expression of TNFRSF1B and promotes osteoclast differentiation. These results reveal the crucial role of miR-125a-5p in the differentiation of osteoclasts.
Highlights
Osteoclasts are derived from monocyte-macrophage precursors that arise from multipotent hematopoietic stem cells [1]
MiR-125a-5p is up-regulated in osteoclastogenesis induced by Colony stimulating factor 1 (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) M-CSF and receptor activator of RANKL are two critical cytokines involved in osteoclast differentiation
tartrate-resistant acid phosphatase (TRAP) staining analysis revealed that RAW 264.7 cells stimulated with M-CSF and RANKL were differentiated into more TRAP-positive osteoclasts than those without M-CSF and RANKL treatment (Fig. 1a)
Summary
Osteoclasts are derived from monocyte-macrophage precursors that arise from multipotent hematopoietic stem cells [1]. Many cytokines and exogenous hormones have been identified to be involved in osteoclastogenesis through transcription factors that positively or negatively modulate osteoclast proliferation, survival, differentiation and function. RANKL induces the expression of nuclear factor of activated T-cells cytoplasmic (NFATc1). MiRNAs contribute to every process of osteogenesis from embryonic bone development to maintenance of tissues, through regulating the growth, differentiation and functional activities of osteoblasts, osteocytes and osteoclasts. Various miRNAs have been reported to regulate osteoblastic differentiation, proliferation and bone formation [6, 7]. The function of miRNAs in osteoclastic differentiation and activity has begun to be elucidated. Recent studies have suggested that miR-125a-3p regulates the expression of G protein-coupled receptor kinase interacting protein 1 (GIT1) and inhibits osteoblastic proliferation and differentiation [13]. The expression and function of miR-125a-5p in osteoclasts remain elusive
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