Mechanically-regulated microRNAs in articular cartilage

Introduction: The role of microRNAs (miRs) in articular cartilage is still not well established, however many studies have reported the differential expression of a number of miRs between healthy and osteoarthritic (OA) articular cartilage. These studies have focused on the OA pathology itself without considering the impact of mechanical load, which is one of the major risk factors implicated in the loss of cartilage integrity and the onset of OA development. Previous studies have already identified a number of mechanically-regulated miRs, therefore I hypothesised that (i) physiological and non-physiological magnitudes of compressive load differentially regulate the expression of mechano-sensitive miRs, and (ii) mechanically-regulated miRs differentially expressed in response to a non-physiological magnitude of load are implicated in the regulation of mechano-sensitive matrix molecule turnover and are involved in OA development. Results: Transcriptional assessment of selected mechanically-regulated matrix molecules demonstrated that loading regimes of 2.5MPa and 7MPa (1Hz, 15 minutes) induced homeostatic and catabolic responses at the gene level respectively, therefore they were selected to represent ‘physiological’ and ‘non-physiological’ magnitudes of loads which have the potential to induce biosynthetic and degradative protein responses if applied for prolonged periods of time. Next generation sequencing (NGS) of articular cartilage miRs libraries demonstrated that the alteration in expression of specific miRs occurs in a magnitude- and time-dependent manner. However, 24h post-load, according to the NGS data, seems to be the most appropriate to observe significant changes in miRs levels. Validation of a few miRs, important for cartilage integrity, at 24h post-load indicated up-regulation of miR-21-5p, miR-27a-5p, miR-221 and miR-222 and down-regulation of miR-483 in response to the ‘non-physiological’ 7MPa magnitude (1Hz, 15 minutes) whereas in explants subjected to a ‘physiological’ 2.5MPa magnitude (1Hz, 15 minutes) the level of these miRs remained unchanged. Identification of target genes of miR-21-5p, miR-221 and miR-222 performed by NGS of RNA extracted from primary articular chondrocytes transfected with specific miR inhibitors demonstrated a number of differentially expressed genes. qPCR validation of these potential miR target genes on RNA collected from cells transfected with functional miR-21-5p, miR-221 and miR-222 inhibitors or mimics identified TIMP-3 as a direct target of miR-21-5p, miR-221 and miR-222, whereas CPEB was targeted by miR-21-5p. Conclusion: This current study confirms the reported mechano-regulation of miR-221 and miR-222, and furthermore demonstrates the novel mechano-regulation of miR-21-5p, miR-27a-5p and miR-483 in cartilage explants. This work is the first to identify TIMP-3 as a target of miR-21-5p and miR-221/-222, and CPEB3 as a direct target of miR-21-5p in primary chondrocytes. An association between the identified differentially-regulated miRs in response to a non-physiological magnitude of load, with those that are expressed in OA cartilage and their regulatory effect on molecules important for cartilage integrity, described in this thesis may pioneer future studies aimed at identifying cartilage biomarkers of load-induced OA and provide therapeutic potential for OA treatment.