First evidences of the role of miR-544a in the modulation of the Wnt signalling in the articular cartilage: potential impact in the pathophysiology of osteoarthritis

Background: Osteoarthritis (OA) is the most common degenerative musculoskeletal condition. Progressive degeneration of the articular cartilage is a hallmark of the disease. A better understanding of OA pathogenetic mechanisms is required for the development of disease-modifying drugs capable of stopping disease progression and promoting cartilage repair. MicroRNAs (miRNAs) constitute a class of small noncoding RNAs with an epigenetic role in the negative regulation of gene expression. The differential expression of several miRNAs between healthy and OA cartilage samples highlighted their implication in the pathogenesis of OA. Several molecular pathways are deregulated in the articular cartilage in OA. The involvement of the Wnt/β-catenin pathway in OA pathogenesis is well known, however its role remains controversial, as both its activation or complete inhibition can lead to cartilage degradation and OA. Previous data showed that miR-544a was the most modulated gene by Wnt3a, a Wnt ligand whose expression and activity is modulated in OA. The purpose of this study was therefore to determine the role of miR-544a in the articular cartilage and in the modulation of the Wnt signalling in this tissue. Methodology: Putative target genes of miR-544a were identified through an in silico analysis. Targets related to the Wnt signalling were then further validated by preparation of target-specific luciferase-based reporter assays. The effect of miR-544a on chondrocyte phenotype was investigated by its transient overexpression in the chondrocyte cell line C28/I2 with or without simultaneous stimulation with recombinant Wnt3a. The expression of chondrocyte phenotypic marker genes was assessed by qPCR while proteoglycan synthesis was measured by alcian blue staining on micromass cultures. The effect of pro-inflammatory cytokines on miR-544a expression was finally investigated by gene expression analysis on primary chondrocytes and C28/I2 cell line. Results: Axin2, CDH1, CTNNBIP1 and GSK-3β were confirmed to be target of miR-544a in chondrocytes. The overexpression of miR-544a in C28/I2 cells up-regulated the expression of Col2a1, Aggrecan and MMP-13 and down-regulated ADAMTS-5 and TIMP-3 at mRNA level. These phenotypic effects did not vary in presence of Wnt3a. MicroRNA-544a did not alter proteoglycan content in our culture conditions. IL-6 and TGF-β up-regulated miR-544a expression levels. Conclusion: Our results showed, for the first time, the possible involvement of miR-544a in the modulation of the Wnt signalling in the articular cartilage as well as in the regulation of chondrocyte metabolism. We can speculate that alterations of miR-544a expression levels, which could be induced by pro-inflammatory cytokines during OA, may contribute to a de-regulated activation of the β-catenin-dependent Wnt signalling exacerbating the catabolic activities of chondrocytes and participating to the pathogenesis of OA.