title: Role of miR-222-3p in c-Src-Mediated Regulation of Osteoclastogenesis.
abstract: MicroRNAs (miRNAs) are small non-coding RNAs that play a mostly post-transcriptional regulatory role in gene expression. Using RAW264.7 pre-osteoclast cells and genome-wide expression analysis, we identified a set of miRNAs that are involved in osteoclastogenesis. Based on in silico analysis, we specifically focused on miR-222-3p and evaluated its role in osteoclastogenesis. The results show that the inhibitor of miR-222-3p upregulated the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and tartrate-resistant acid phosphatase (TRAP), while its mimicking agent downregulated their mRNA levels. Western blot analysis showed that its inhibitor increased the protein levels of TRAP and cathepsin K, while its mimicking agent decreased their levels. Genome-wide mRNA expression analysis in the presence and absence of receptor activator of nuclear factor κ-B ligand (RANKL) predicted c-Src as a potential regulatory target of miR-222-3p. Live cell imaging using a fluorescence resonance energy transfer (FRET) technique revealed that miR-222-3p acted as an inhibitor of c-Src activity, and a partial silencing of c-Src suppressed RANKL-induced expression of TRAP and cathepsin K, as well as the number of multi-nucleated osteoclasts and their pit formation. Collectively, the study herein demonstrates that miR-222-3p serves as an inhibitor of osteoclastogenesis and c-Src mediates its inhibition of cathepsin K and TRAP.
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA. stakigaw@iupui.edu.|Department of Orthopaedic Surgery, Mie University Graduate School of Medicine, Mie 514-8507, Japan. stakigaw@iupui.edu.
authors.forename: Shinya
authors.initials: S
authors.lastname: Takigawa
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA. andychen@umail.iu.edu.
authors.forename: Andy
authors.initials: A
authors.lastname: Chen
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA. wanq@purdue.edu.
authors.forename: Qiaoqiao
authors.initials: Q
authors.lastname: Wan
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA. sungna@iupui.edu.
authors.forename: Sungsoo
authors.initials: S
authors.lastname: Na
authors.affiliation: Department of Orthopaedic Surgery, Mie University Graduate School of Medicine, Mie 514-8507, Japan. a-sudou@clin.medic.mie-u.ac.jp.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Sudo
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA. hyokota@iupui.edu.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Yokota
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA. hamak@dpc.agu.ac.jp.|Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, Nagoya 464-8650, Japan. hamak@dpc.agu.ac.jp.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Hamamura
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D001862:Bone Resorption / Q000235:genetics* / Q000378:metabolism*; D000081247:CSK Tyrosine-Protein Kinase; D056657:Cathepsin K / Q000378:metabolism; D002460:Cell Line; D016000:Cluster Analysis; D004789:Enzyme Activation; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D020868:Gene Silencing; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D050778:NFATC Transcription Factors / Q000378:metabolism; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; C546123:MIRN222 microRNA, mouse; D035683:MicroRNAs; D050778:NFATC Transcription Factors; D053245:RANK Ligand; D012333:RNA, Messenger; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D056657:Cathepsin K
keywords: RAW264.7 cells; c-Src; miRNA; microarray; osteoclastogenesis