Regulation of microRNA-221, -222, -21 and -27 in articular cartilage subjected to abnormal compressive forces.

KEY POINTS microRNAs (miRs) are small non-coding molecules that regulate post-transcriptional target gene expression. miRs are involved in regulating cellular activities in response to mechanical loading in all physiological systems, but it is largely unknown whether this response differs with increasing magnitudes of load. miR-221, miR-222, miR-21-5p, and miR-27a-5p were significantly increased in ex vivo cartilage explants subjected to increasing load magnitude and in in vivo joint cartilage exposed to abnormal loading. TIMP3 and CPEB3 are putative miR targets in chondrocytes Identification of mechanically regulated miRs that have potential to impact on tissue homeostasis provides a mechanism by which load-induced tissue behaviour is regulated, both in health and pathology, in all physiological systems. ABSTRACT Objective microRNAs (miRs) are small non-coding molecules that regulate post-transcriptional target gene expression and are involved in mechano-regulation of cellular activities in all physiological systems. It is unknown whether such epigenetic mechanisms are regulated in response to increasing magnitudes of load. This study investigated mechano-regulation of miRs in articular cartilage subjected to 'physiological' and 'non-physiological' compressive loads in vitro as a model system and validated findings in an in vivo model of abnormal joint loading. Design Bovine full-depth articular cartilage explants were loaded to 2.5MPa (physiological) or 7MPa (non-physiological) (1Hz, 15 minutes) and mechanically-regulated miRs identified using Next Generation Sequencing and verified using quantitative PCR. Downstream targets were verified using miR-specific mimics or inhibitors in conjunction with 3'-UTR luciferase activity assays. Results A subset of miRs were mechanically-regulated in ex vivo cartilage explants and in vivo joint cartilage. miR-221, miR-222, miR-21-5p, and miR-27a-5p were increased and miR-483 levels decreased with increasing load magnitude. Tissue Inhibitor of Metalloproteinase 3 (TIMP3) and Cytoplasmic Polyadenylation Element Binding Protein 3 (CPEB3) were identified as putative downstream targets. Conclusion Our data confirms miR-221 and -222 mechano-regulation and demonstrates novel mechano-regulation of miR-21-5p and miR-27a-5p in ex vivo and in vivo cartilage loading models. TIMP3 and CPEB3 are putative miR targets in chondrocytes. Identification of specific miRs that are regulated by increasing load magnitude, and their potential to impact on tissue homeostasis, has direct relevance to other mechano-sensitive physiological systems and provides a mechanism by which load-induced tissue behaviour is regulated, both in health and pathology. This article is protected by copyright. All rights reserved.