Project description:Objective: To Identify gene changes in the medial tibial plateau of articular cartilage at 2, 4 and 8 weeks after destabilisation of the medial meniscus (DMM) in mice and compare our data set with previously published sets to ascertain dysregulated pathways and genes in osteoarthritis. Materials and methods: RNA was extracted from the ipsilateral and contralateral medial tibial plateaus, amplified, labelled and hybridized on Illumina WG_v2 microarrays. Results were confirmed by RT-PCR for selected genes. Results: Transcriptional analysis and network reconstruction revealed changes in extracellular matrix (ECM) and cytoskeletal genes induced by DMM. TGFalpha signalling pathway and complement and coagulation cascade genes were regulated at 2 weeks. Fibronectin (FN1) is a hub in a reconstructed network of potential protein-protein interactions at 2 weeks. Regulated genes decrease over time and by eight weeks fibromodulin (FMOD) and tenascin N (TNN) are the only dysregulated genes present in the DMM operated knees. Comparison with human and rodent published gene sets discovered genes overlapping between our array and 6 others. Conclusions: Cartilage contributes a minute percentage to the RNA extracted from the whole joint (>0.2%), yet is sensitive to changes in gene expression post-DMM. The post-DMM transcriptional reprogramming wanes over time and dissipates by 8 weeks. Common pathways between published gene sets include focal adhesion, regulation of actin cytoskeleton and TGFalpha. Common genes include Jagged 1 (Jag1), Tetraspanin 2 (Tspan2), neuroblastoma, suppression of tumourigenicity 1 (Nbl1) and N-myc downstream regulated gene 2 (Ndrg2). The concomitant genes and pathways we identify are novel and warrant further investigation as biomarkers or modulators of osteoarthritis. In total 18 samples. Each sample was a pool of the medial tibial plateau of 8 mice. Two categories (Treated and untreated) with three biological replicates each and three time points (2, 4 and 8 weeks post-surgery).

Project description:Objective Animal models of post-traumatic osteoarthritis (PTOA) recapitulate the pathological changes observed in human PTOA. Here we aimed to compare the cartilage transcriptome responses of a non-surgical, mechanically induced rupture of the anterior cruciate ligament (ACL) model and the surgical destabilisation of the medial meniscus (DMM) model. Methods Skeletally mature male C57Bl6 mice were subjected to either the non-surgical, mechanical ACL rupture or surgical DMM models and transcriptome profiling performed on micro-dissected cartilage at day 7 and 42 post-procedure, respectively; in general, naïve animals served as controls. MicroRNA profiling was also performed on the ACL rupture model. Expression levels of a miRNA of interest, miR-199-5p, were inhibited in primary human articular chondrocytes (HAC) with RNA-seq and 3’UTR assays used to identify and valid potential target genes. Results The number of differentially expressed genes between the two models were comparable and highly correlative (Spearman R =0.8, P<2.2E-16). Gene ontology enrichment analysis identified similarly enriched pathways, containing anabolic terms including ‘extracellular matrix organisation’ enriched for the upregulated genes. Within the ACL rupture miRNA transcriptome, miR-199-5p family members were amongst the most abundantly, and differentially expressed, which was replicated in the DMM cartilage by qRT-PCR. Inhibition of miR-199-5p in HAC led to a comparable transcriptome response to that observed in both human OA damaged vs intact cartilage and murine DMM cartilage datasets. Several genes, including GIT1, NCEH1, SOS2 and ECE1 were all experimentally verified as targets. Conclusion For the first time, we have characterised both the mRNA and miRNA articular cartilage signature in the ACL rupture model and demonstrated highly correlative responses with the DMM PTOA model. These data support the use of the ACL rupture model as a non-invasive alternative to DMM.

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Medial Tibial Plateau articular cartilage changes 2 ,4 and 8 weeks after DMM

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