| Summary: | Osteoarthritis (OA) is a debilitating disease suffered by millions of people around the world. It affects the whole joint but is defined in part by a degradation of cartilage which becomes more pronounced as the disease progresses. The initiation and progression of OA is still not fully characterised. MicroRNAs are small, non-coding RNA molecules which are known to regulate gene expression, however, at the moment it is unclear exactly how many may be expressed in cartilage or how their expression may alter during osteoarthritis. Using human OA cartilage and cells derived from it, we have optimised small RNA purification and have shown that there is a clear difference between microRNA expression in cartilage compared with cell isolates, with miR-140 most highly expressed in cells immediately after digestion. This may suggest a stress-type response. RNA purified from chondrocytes immediately after digestion from cartilage was utilised for Illumina GIIX deep sequencing in an attempt to identify novel microRNAs in addition to expressed known microRNAs. 990 previously documented and 1621 potential novel microRNAs were discovered and 16 novel candidates were chosen for further validation. Validation was carried out using northern blotting and qRT-PCR on a number of samples and tissue types. In addition, the selected candidates were also examined in chondrogenesis models, and one candidate was probed in mouse embryo in situ hybridisation in an attempt to localise the candidate to cartilage tissue. Three of the candidates with the most promising validation results were analysed for function using a whole genome microarray combined with computational target search analysis. A number of likely targets obtained from the array were further validated by subcloning and luciferase assays, resulting in the discovery of ITGA5 as a target of candidate novel 11. The outcomes from this research provide us with an increased understanding of the miRNome of human OA cartilage and could impact upon future drug development and the use of microRNAs as biomarkers.
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