THU0076 Microrna-124 Inhibits Progression of Adjuvant-Induced Arthritis in Rats

Background MicroRNAs (miRNAs) are small (∼22 nt) endogenous, noncoding RNAs that act as post-transcriptional regulators of gene expression.[1] We previously reported that miR-124a expression decreases proliferative RA synoviocytes as compared to osteoarthritis synoviocytes. The transfection of precursor -miR-124a into RA synoviocytes significantly suppresses synoviocyte proliferation and suppresses the secretion of monocyte chemoattractant protein 1 (MCP-1). Furthermore, miR-124a directly downregulates the production of cyclin-dependent kinase 2 (CDK-2) and MCP1 proteins at translational level. [2] Objectives The aim of this study is to investigate the in vivo effect of miRNA-124 (miR-124, the rat analog of human miR-124a) on adjuvant-induced arthritis (AIA) in rats. Methods 1) Experimental arthritis was induced in rats by injecting 0.1 ml of incomplete Freund9s adjuvant (IFA) and 1.0 mg of a heat-killed Mycobacterium tuberculosis (Difco), into the base of the tail on day 0. On day 9, Pre-miR-124 (Life Technologies) or Pre-miR Negative Control #2 (Life Technologies) was mixed with AteloGene Reagent (Koken), and 50μl of the mixture (14.3 pmol/μl of miRNA) was injected into rats at the right ankle joints. The severity of arthritis was observed from day 1 to day 18. Rats were sacrificed on day 18. The hind feet were used for micro-computed tomography (micro CT) analysis, and the ankles were used for both histopathological analysis and quantitative real-time RT-PCR. For western blotting analysis, rats were sacrificed on day 12, and the ankle tissues were collected for protein extraction. 2) For luciferase reporter assay, the 39-UTR of mRNAs and their site-directed mutants were PCR-amplified, and cloned into psiCHECK-2 vector (Promega). Pre-miRNAs were transfected into E11 synovial fibroblast cell line, and the cells were transfected with these vectors on the next day. After 48 h, the cells were lysed and luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer9s instructions. 3) The effect of pre-miR-124 or pre-miR-124a on the differentiation of human osteoclasts was examined by tartrate-resistant acid phosphatase (TRAP) staining. Results 1) MiR-124 suppressed AIA in rats, as demonstrated by decreased synoviocyte proliferation, leukocyte infiltration, and cartilage/ bone destruction at ankle. Osteoclast counts and expression level of RANKL, ITGB1 and NFATc1 were reduced in AIA rats treated with pre-miR-124 at ankle. 2) Luciferase analysis showed that miR-124 directly targeted the 39-UTR of the rat NFATc1, ITGB1, SP1, and CEBPA mRNAs. Pre-miR-124 also suppressed NFATc1 expression in RAW264.7 cells. 3) Both miR-124 and miR-124a directly targeted the 39-UTR of human NFATc1 mRNA, and both pre-miR-124 and pre-miR-124a suppressed the differentiation of human osteoclasts. Conclusions We showed that miR-124 ameliorated AIA by suppressing critical prerequisites for arthritis development, such as RANKL and NFATc1. Thus, miR-124a is a candidate for therapeutic use for human rheumatoid arthritis. References Chu CY et al. J Cell Physiol 2007:412-9. Nakamachi Y et al. Arthritis Rheum 2009; 1294-304. Disclosure of Interest None declared