Transcriptomic analysis of post-traumatic osteoarthritis mouse models identifies miR-199a-5p as a regulator of abnormal mechanical loading in articular cartilage (RNA-Seq)
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. Overall design: Wild-type C57bl6 mice were used.  On the injured mice the the anterior cruciate ligament (ACL) rupture model was performed on the  right leg. The contralateral left leg acts as a control. mice were sacrifised at 1 or 7 day post-injury. Mice naive to treatment (day_7_naive) also acted as a further control. Medial knee cartilage caps were dissected, washed with PBS and frozen in liquid nitrogen and RNA isolated using TRIzol® reagent according to manufacturer's protocol (ThermoFisher Scientific). Total RNA and miRNA were purified from the TRIzol®-cartilage mixture using a mirVana™ miR Isolation Kit followed by DNAse treatment (Ambion, Fisher Scientific, Loughborough, UK) following the manufacturer's protocol   For each sample cartilage is pooled from 4 mice/treatment.  Breifly. for the ACL rupture, mice were anaesthetized with isoflurane and custom-built cups used to hold the right ankle and knee in flexion with a 30o offset prior to the application of a 0.5 N pre-load (ElectroForce3200; TA Instruments, Elstree, UK). A single 12 N load at a velocity of 1.4 mm s-1 was then applied resulting in anterior cruciate ligament (ACL) rupture; mechanical loading was always conducted in the morning. Buprenorphine (0.05 mg kg-1) was administered subcutaneously to mice at the start of the experiment; animals were able to move freely and were monitored for welfare and lameness until termination of the experiment by cervical dislocation.