MiRNA-340 inhibits osteoclast differentiation via repression of MITF.

Hongying Zhao,Yazeng Huang,Haiyu Shao,Mengran Jin,Jianwen Liu,Jun Zhang,Jinping Chen

doi:10.1042/bsr20170302

Abstract

Many miRNAs play critical roles in modulating various biological processes of osteoclast differentiation and function. Microphthalmia-associated transcription factor (MITF), a target of miR-340, served as pivotal transcription factor involved in osteoclast differentiation. However, the role of miR-340 and MITF during osteoclast differentiation has not yet been clearly established. Tartrate-resistant acid phosphatase (TRAP) staining assay was performed to identify osteoclasts differentiated from bone marrow-derived macrophages (BMMs). Quantitative reverse transcription PCR (qRT-PCR) or Western blotting was undertaken to examine the mRNA or protein expression respectively. Luciferase reporter assay was performed to investigate the interaction between miR-340 and MITF. MITF was knocked down and miR-340 was overexpressed and transfected into BMMs to detect their effects on osteoclast differentiation. Firstly, qRT-PCR analysis showed that miR-340 was down-regulated during osteoclast differentiation stimulated by macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB (RANK) ligand (RANKL). Besides, we found that overexpression of miRNA-340 inhibited osteoclast differentiation and suppressed both the mRNA and protein level of MITF. Finally, Western blot and qRT-PCR analysis revealed that silencing MITF inhibited TRAP, calcitonin receptor, V-ATPase d2, and cathepsin K. miR-340 suppresses osteoclast differentiation by inhibiting MITF. Our findings may provide promising therapeutic targets for osteoclast-associated diseases.

Highlights

Osteoclasts, responsible for bone resorption, are multinucleated cells (MNCs) differentiated from macrophage/monocyte precursors [1,2]
Tartrate-resistant acid phosphatase (TRAP) staining analysis revealed that bone marrow-derived macrophage (BMM) stimulated with macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) were differentiated into more TRAP-positive osteoclasts than that without treatment of M-CSF and RANKL (Figure 1A), indicating treatment with M-CSF, and RANKL may serve as osteoclastogenesis condition
Statistical analysis of Quantitative reverse transcription PCR (qRT-PCR) demonsrated that relative expression level of miR-340 in BMMs treated with M-CSF and RANKL was down-regulated in a time-dependent manner

Summary

Introduction

Osteoclasts, responsible for bone resorption, are multinucleated cells (MNCs) differentiated from macrophage/monocyte precursors [1,2]. It is widely known that bone homeostasis is dependent on the balance between bone resorption mediated by osteoclasts and bone formation by osteoblasts [3]. Excessive bone resorption induced by overactivity of osteoclasts is often associated with bone loss-related diseases including osteoporosis, rheumatoid arthritis, periodontal disease, multiple myeloma, and metastatic cancers [4,5]. Recent studies have reported that many miRNAs have been identified to involve in bone metabolism and osteoclast differentiation [8]. Ma et al [13] has presented evidence that miR-340 was down-regulated during osteoclast differentiation using microarray analysis, which was associated with the progression of osteoporosis. Little literature concerning the precise role of miR-340 implicated in osteoclastogenesis has been published

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Results

Conclusion