LAUSR

Background Osteoarthritis (OA), the most common type of joint disease, is characterised by progressive and irreversible degradation of articular cartilage. Dysregulated gene expression has also been linked to disease pathogenesis. Lin28A is an evolutionarily conserved RNA binding protein and known to play a critical role in development, metabolism and tumorigenesis. However, the role of Lin28A in osteoarthritis is not yet explored. Objectives To study the role of Lin28A in the regulation of genes associated with OA pathogenesis in human chondrocytes. Methods Primary human chondrocytes were isolated from the undamaged portion of the knee OA cartilage by enzymatic digestion and were cultured in DMEM/F12% and 10% FCS. Total RNA from cartilage explants or chondrocytes was prepared using Trizol and was made DNA-free by on-column digestion method. mRNA expression levels of Lin28A, Lin28B, MMP-13, IL-6, COX2 and iNOS were quantified by TaqMan assays. Protein expression was analysed by immuno-blotting using validated antibodies. Nucleofection was used for the siRNA mediated depletion or plasmid mediated overexpression of Lin28A gene was used to study its role in chondrocyte function under pathological conditions. RNA immunoprecipitation (RIP) with anti-Lin28A antibody was performed to identify the interacting mRNA partners. Stability of the mRNAs was determined by Actinomycin-D chase experiments. Results Human OA cartilage samples (n=6) analysed were found to express Lin28A but not Lin28B transcripts. Lin28A mRNA expression was significantly high in the damaged OA cartilage compared to the smooth cartilage from the same patient (n=3, p<0.05). Stimulation with IL-1β induced the high levels of Lin28A mRNA and protein expression in OA chondrocytes in a time dependent as well as dose dependent manner. siRNA mediated depletion of Lin28A expression in OA chondrocytes inhibited the IL-1β-induced expression of MMP-13, IL-6, COX2 and iNOS mRNAs. Importantly, the overexpression of Lin28A induced the expression of MMP-13, IL-6, COX-2 and iNOS mRNA and protein in OA chondrocytes. Immunoblotting analysis showed that the Lin28A depleted OA chondrocytes treated with IL-1β also produced significantly low levels of IL-6, MMP-13 and COX-2 protein compared to chondrocytes transfected with scrambled siRNAs (n=3, p<0.05). RIP analyses in IL-1β treated OA chondrocytes with anti-Lin28A antibody revealed that the MMP-13, IL-6 and COX2 mRNAs were pulled down with anti-Lin28A and were highly enriched when compared with the mRNA population pulled down by isotype control antibody. siRNA mediated depletion of Lin28A expression resulted in decreased half-life of IL-6 and COX-2 mRNAs, while the overexpression of Lin28A had the opposite effect in IL-1β stimulated OA chondrocytes. This indicated that Lin28A contributes towards the stability of IL-6 and COX-2 mRNAs. Conclusions Our data for the first time demonstrate that Lin28A plays a key role in OA pathogenesis by stabilising the expression of catabolic gene transcripts in human chondrocytes under pathological conditions. These data revealed a previously unidentified role of Lin28A in chondrocytes and identify it as a potential therapeutic target for the treatment of OA. Acknowledgements Supported by NIH grants RO1 AT-007373, RO1 AR-067056 and funds from the Northeast Ohio Medical University to TMH. Disclosure of Interest I. Ahmad: None declared, M. Ansari: None declared, M. Khan: None declared, T. Haqqi Grant/research support from: USPHS Grants