MicroRNA-20a regulates autophagy related protein-ATG16L1 in hypoxia-induced osteoclast differentiation |
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| Affiliation: | 1. Graduate Institute of Clinical Medical Science, China Medical University, No. 91 Hsueh-Shih Rd., Taichung, Taiwan;2. Department of Pediatric Dentistry, China Medical University Hospital, No. 2 Yu-Der Rd., Taichung, Taiwan;3. School of Dentistry, China Medical University, No. 91 Hsueh-Shih Rd., Taichung, Taiwan;4. Department of Oral & Maxillofacial Surgeon, China Medical University Hospital, No. 2 Yu-Der Rd., Taichung, Taiwan;5. Division of Nephrology, Department of medicine, China Medical University Hospital, No. 2 Yu-Der Rd., Taichung, Taiwan;6. Division of Cardiology, Department of medicine, China Medical University Hospital, No. 2 Yu-Der Rd., Taichung, Taiwan;7. Department of Anesthesiology, China Medical University Hospital, No. 2 Yu-Der Rd., Taichung, Taiwan;1. Primary Health Care Center Eap Sardenya, Biomedical Research Institute Sant Pau (IIB Sant Pau), Sardenya 466, Barcelona 08025, Spain;2. Institut Universitari d''Investigació en Atenció Primària Jordi Gol (IDIAP Jorid Gol), Gran Vía Corts Catalanes 587, 08007 Barcelona, Spain. Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain;3. Research Unit, Family Medicine, Girona, Jordi Gol Institute for Primary Care Research (IDIAP Jordi Gol), Maluquer Salvador 11, Girona 17002, Spain;4. TransLab Research Group, Department of Medical Sciences, School of Medicine, University of Girona, Emili Grahit 77, Girona 17003, Spain;5. Primary Care Services Information System, Catalan Health Institute (ICS), Gran Via Corts Catalanes 587, Barcelona 08007, Spain;6. Primary Care Services, Girona, Catalan Institute of Health (ICS), Maluquer Salvador 11, Girona 17003, Spain;7. Musculoskeletal Research Unit and RETICEF, IMIM Research Foundation, Parc de Salut Mar and Instituto de Salud Carlos III, Doctor Aiguader 88, Barcelona 08003, Spain;8. MRC Lifecourse Epidemiology Unit, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, United Kingdom;9. Oxford NIHR Musculoskeletal Biomedical Research Unit, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Windmill Road, Oxford OX3 7HE, United Kingdom;1. Department of Therapeutic Radiology, University of Minnesota, MN, USA;2. Masonic Cancer Center, University of Minnesota, MN, USA;3. Department of Obstetrics and Gynecology, University of Minnesota, MN, USA;4. Mindways Software Inc., Austin, TX, USA;5. Center for Magnetic Resonance Research, Department of Radiology, University of Minnesota, MN, USA;6. Biostatistics Core, Masonic Cancer Center, University of Minnesota, MN, USA;7. Department of Medicine, Division of Rheumatology, University of Wisconsin, Madison, USA;8. Department of Medicine, University of Minnesota, MN, USA;9. Family Medicine and Community Health, University of Minnesota, MN, USA;10. Campus Bio-Medico University, School of Medicine, Rome, Italy;11. Department of Biostatistics, University of Minnesota, Minneapolis, USA;12. Maine Medical Center Research Institute, Scarborough, ME, USA;1. Institute of Naval Medicine, Alverstoke, Gosport PO12 2DL, UK;2. Department of Nutritional Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK;3. Bone Biology Laboratory, University College London, London WC1E 6BT, UK;4. The Osteoporosis Centre, University Hospital Southampton NHS Foundation Trust, Southampton, Hampshire, UK;5. MRC Lifecourse Epidemiology Unit, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK;1. INSERM UMR 1033, Equipe Qualité Osseuse et Marqueurs Biologiques, Lyon, France;2. Université de Lyon, Lyon, France;3. ENT Department, Hôpital Edouard Herriot, Lyon, France;4. Lyon Neurosciences Research Center, Brain Dynamics and Cognition Team, CRNL, INSERM UMR 1028, CNRS UMR 5292, Lyon, France;1. Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité — Universitätsmedizin Berlin, 13353 Berlin, Germany;2. Berlin-Brandenburg School for Regenerative Therapies (BSRT), Charité — Universitätsmedizin Berlin, 13353 Berlin, Germany;3. Julius Wolff Institute and Center for Musculoskeletal Surgery, Charité — Universitätsmedizin Berlin, 13353 Berlin, Germany;4. Institute for Medical Genetics and Human Genetics, Charité — Universitätsmedizin Berlin, 13353 Berlin, Germany;5. Research Group Development and Disease, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany;6. Biopharm GmbH, 69115 Heidelberg, Germany |
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| Abstract: | Autophagy and autophagy-related proteins (ATGs) play decisive roles in osteoclast differentiation. Emerging lines of evidence show the deregulation of miRNA in autophagic responses. However, the role of hypoxia and involvement of miRNA in osteoclast differentiation are unclear. In the present study, we demonstrate that hypoxia caused induction of autophagy and osteoclast differentiation markers in RAW264.7 cells stimulated with M-CSF and RANKL. In addition, miR-20a was significantly repressed during hypoxia and identified as the prime candidate involved in hypoxia-induced osteoclast differentiation. The results from dual luciferase reporter assay revealed that miR-20a directly targets Atg16l1 by binding to its 3′UTR end. Further, miR-20a transfection studies showed significant down regulation of autophagic proteins (LC3-II and ATG16L1) and osteoclast differentiation markers (Nfatc1, Traf6, and Trap) thus confirming the functional role of miR-20a under hypoxic conditions. Results of chromatin immunoprecipitation assay showed that HIF-1α binds to miRNA-20a. From miRNA Q-PCR results, we confirmed that shRNA HIF-1α knockdown significantly downregulated both autophagy (LC3, p62, Atg5, Atg12, Atg16l1, Atg7, Becn1, Atg9a) and osteoclast markers (Traf6, Nfatc1, Ctsk, cFos, Mmp9, Trap) in RAW264.7 cells. Thus, our findings suggest that the regulatory axis of HIF-1α-miRNA-20a-Atg16l1 might be a critical mechanism for hypoxia-induced osteoclast differentiation. |
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