Swingler, T. E. | Search bioRxiv

bioRxiv2024

microRNA-544a as a new modulator of the Wnt-signalling network in the articular cartilage and osteoarthritis

ObjectiveDetermining the effect of microRNA-544a (miR-544a) in articular chondrocytes isolated from patients affected by osteoarthritis (OA) and its role in the modulation of the Wnt signalling. MethodsArticular chondrocytes were isolated from patients undergoing joint replacement because of OA. Expression levels of miR-544a were measured by PCR and by in situ hybridization. Putative targets of miR-544a were confirmed by reporter assay and by qPCR in cells stimulated with a miR-544a mimic. The effect of miR-544a on chondrocyte metabolism was monitored by qPCR for phenotypic markers, protein expression levels of aggrecan neoepitopes/MMP-13 and modulation of alcian blue content in micromass cultures, upon stimulation with a miR-544a mimic. The expression levels of MMP-13 and Aggrecan neoepitopes in response to miR-544a stimulation was also measured in co-stimulation with Xav-939 and KN93, which are respectively {beta}-catenin and CaMKII inhibitors. ResultsOur results suggest that miR-544a enhances the activation of the Wnt-signalling in the articular chondrocytes, by downregulating the expression of components of the Wnt/{beta}-catenin destruction complex. The expression of miR-544a is higher in chondrocytes isolated from damaged areas of the articular cartilage removed from OA patients, and can be upregulated by pro-inflammatory and pro-fibrotic cytokines. miR-544a exerts a pro-catabolic effect of articular chondrocytes, which is rescued both by the inhibition of the Wnt/{beta}-catenin and Wnt/CaMKII signalling pathways. Conclusionour results point to miR-544a as a new, important modulator of the Wnt signalling network within the articular cartilage suggesting a key role for microRNAs in regulating how the multiple branches of the network and their interaction modulate cartilage homeostasis.

pharmacology and toxicology↗

bioRxiv2020

microRNA-seq of cartilage reveals an over-abundance of miR-140-3p which contains functional isomiRs

MiR-140 is selectively expressed in cartilage. Deletion of the entire miR-140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology, however the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5 and 3 end, with >99% having one of two seed sequences (5 bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3 UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published datasets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.

molecular biology↗