miR-324 mediates bone homeostasis through the regulation of osteoblast and osteoclast differentiation and activity

miR-324 mediates bone homeostasis through the regulation of osteoblast and osteoclast differentiation and activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article miR-324 mediates bone homeostasis through the regulation of osteoblast and osteoclast differentiation and activity David Young, Dan Hayman, Francesca Johnson de Sousa Brito, Hua Lin, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3706432/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract microRNAs (miRNAs) modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. miR-324 is implicated in bone and cartilage maintenance, defects of which result in the common age-related diseases osteoporosis and osteoarthritis. Here, in global miR-324-null mice cartilage damage was increased in both surgically and ageing-induced osteoarthritis, despite minimal changes to the cartilage transcriptome. However, in vivo micro-computed tomography and histology demonstrated that the mice showed increased bone mineral density and both trabecular and cortical thickness, with effect magnitudes increasing with age. The bone marrow of miR-324-null mice also had reduced lipid content while in vivo TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation. Ex vivo assays showed that the high bone mass phenotype of miR-324-null mice resulted from increased osteoblast activity and decreased osteoclastogenesis. RNA-seq analysis of osteoblasts, osteoclasts and bone marrow macrophages and target validation assays identified that the osteoclast fusion regulator Pin1 and the master osteogenic regulator were targets of miR-324-5p in osteoclast lineage cells and osteoblasts, respectively. Indeed, in vitro Runx2 overexpression recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo by the loss of miR-324. Overall, these data demonstrate the importance of miR-324 in skeletal biology and that altered bone homeostasis is likely causal for the increased cartilage damage observed during osteoarthritis and ageing. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis. Health sciences/Diseases/Endocrine system and metabolic diseases/Metabolic bone disease/Osteopetrosis Biological sciences/Physiology/Bone Full Text Additional Declarations (Not answered) Supplementary Files Supplementaldatatable1.csv Dataset 1 Supplementaldatatable2.csv Dataset 2 Supplementaldatatable3.csv Dataset 3 SupplementaryData.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3706432","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":256241771,"identity":"18ee8c68-77ff-4ab4-9492-737cdfeb6aa7","order_by":0,"name":"David Young","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYDCCAzAGewMDwwMwi41YLTxAVgJpWiQSiNTCd4D5ATNPxeE8+ci3Bz8kMNjJM0ikJeDVInmAzYCZ58zhYsPbeclAi5INGyTSDuDVYnCAwYCZt+1w4sbZOWZAhzEnMEikNxDQwv4BomXmGZCWemK08EBsmS/BA9JyGKiFgMMkD/MUHJxzJj1xA0+OsUSCwXHDNp5nCXi18B1v3/jgTYV14vz2M4YfPlRUy/Ozpxng1cLAzMBwiAfsQrA7CUckGDD+ABLyDcQoHQWjYBSMghEJAB4QQk0f3JSUAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-7078-6745","institution":"Newcastle University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Young","suffix":""},{"id":256241772,"identity":"78461d54-7326-4c2c-abaa-d4b6f13956a4","order_by":1,"name":"Dan Hayman","email":"","orcid":"","institution":"Newcastle University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Hayman","suffix":""},{"id":256241773,"identity":"485b7adf-06db-4642-9eaa-d123deaf446b","order_by":2,"name":"Francesca Johnson de Sousa Brito","email":"","orcid":"https://orcid.org/0000-0001-9376-0487","institution":"University of Liverpool","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"Johnson de Sousa","lastName":"Brito","suffix":""},{"id":256241774,"identity":"c18ba1a2-e14e-4bfe-b3e9-986d2fce09c5","order_by":3,"name":"Hua Lin","email":"","orcid":"","institution":"Newcastle University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Lin","suffix":""},{"id":256241775,"identity":"b18d6f5f-2006-4a42-b541-bf252182c681","order_by":4,"name":"Amanda Prior","email":"","orcid":"","institution":"University of Liverpool","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amanda","middleName":"","lastName":"Prior","suffix":""},{"id":256241776,"identity":"d2927712-8f9e-4369-87c4-74e3452ac4df","order_by":5,"name":"Gemma Charlesworth","email":"","orcid":"","institution":"University of Liverpool","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gemma","middleName":"","lastName":"Charlesworth","suffix":""},{"id":256241777,"identity":"f31f2013-7102-4a76-8e3c-8dcea67a9fab","order_by":6,"name":"Yao Hao","email":"","orcid":"","institution":"First Hospital of Shanxi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Hao","suffix":""},{"id":256241778,"identity":"e91c0d0c-acd7-401f-bfb8-ae65b9080335","order_by":7,"name":"Rachel Pearson","email":"","orcid":"","institution":"Newcastle University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rachel","middleName":"","lastName":"Pearson","suffix":""},{"id":256241779,"identity":"9c7a8fb7-69c1-4e6d-8060-d75c6a5d980d","order_by":8,"name":"Jamie Soul","email":"","orcid":"","institution":"Newcastle University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jamie","middleName":"","lastName":"Soul","suffix":""},{"id":256241780,"identity":"9bfb4a96-f1d9-4e84-bd3b-02384900380d","order_by":9,"name":"Ian Clark","email":"","orcid":"","institution":"University of East Anglia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ian","middleName":"","lastName":"Clark","suffix":""},{"id":256241781,"identity":"7e33eea2-49fd-4920-ac44-ccaee54a69f1","order_by":10,"name":"Katarzyna Piróg","email":"","orcid":"","institution":"Newcastle University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Katarzyna","middleName":"","lastName":"Piróg","suffix":""},{"id":256241782,"identity":"1dd8496a-edec-42c5-857e-2b2cb780de17","order_by":11,"name":"Rob van 't Hof","email":"","orcid":"","institution":"University of Liverpool","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rob","middleName":"van 't","lastName":"Hof","suffix":""},{"id":256241783,"identity":"c3da60b7-88f4-4966-a0d6-11d1e8d683cd","order_by":12,"name":"Matt J. 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One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. miR-324 is implicated in bone and cartilage maintenance, defects of which result in the common age-related diseases osteoporosis and osteoarthritis. Here, in global miR-324-null mice cartilage damage was increased in both surgically and ageing-induced osteoarthritis, despite minimal changes to the cartilage transcriptome. However, \u003cem\u003ein vivo\u003c/em\u003e micro-computed tomography and histology demonstrated that the mice showed increased bone mineral density and both trabecular and cortical thickness, with effect magnitudes increasing with age. The bone marrow of miR-324-null mice also had reduced lipid content while \u003cem\u003ein vivo\u003c/em\u003e TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation. \u003cem\u003eEx vivo\u003c/em\u003e assays showed that the high bone mass phenotype of miR-324-null mice resulted from increased osteoblast activity and decreased osteoclastogenesis. RNA-seq analysis of osteoblasts, osteoclasts and bone marrow macrophages and target validation assays identified that the osteoclast fusion regulator Pin1 and the master osteogenic regulator were targets of miR-324-5p in osteoclast lineage cells and osteoblasts, respectively. Indeed, \u003cem\u003ein vitro\u003c/em\u003e Runx2 overexpression recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed \u003cem\u003ein vivo\u003c/em\u003e by the loss of miR-324. Overall, these data demonstrate the importance of miR-324 in skeletal biology and that altered bone homeostasis is likely causal for the increased cartilage damage observed during osteoarthritis and ageing. 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