Background/Purpose: Articular bone erosion in rheumatoid arthritis (RA) is a consequence of synovial inflammation that leads to disability for patients. Cells within the synovium secrete inflammatory cytokines, RANKL, and Wnt signaling inhibitors, promoting osteoclastogenesis and inhibiting osteoblast function and bone formation. MicroRNAs (miRNAs) are key regulators of skeletal remodeling and also play a role in RA pathogenesis. Therefore, we hypothesized that miRNAs derived from synovial tissues may regulate the erosive process in RA at least in part through effects on osteoblasts.

Methods: To address whether synovium-derived miRNAs regulate osteoblast differentiation and bone formation, we used the serum transfer model of RA. We performed Fluidigm high-throughput expression profiling of 750 miRNAs in pooled synovial samples from non-arthritic and arthritic mice. We also performed gene array (Affymetrix) analysis in these same RNA samples and compared gene expression and miRNA expression from array data. To identify miRNAs regulating skeletal pathways, we collected target genes of up-regulated and down-regulated miRNAs using TargetScan. We restricted downstream analysis to targets differentially expressed by gene array. Finally, we transfected a selected miRNA in murine calvarial osteoblasts to examine its role in cellular differentiation.

Results: Our comparative analysis identified 417 up-regulated genes that are predicted targets of 72 down-regulated miRNAs (1.5 fold or >), and 536 down-regulated genes that are predicted targets of 59 up-regulated miRNAs. Gene ontology analysis of the miRNA-targeted genes revealed significant enrichment of skeletal pathways involved in osteoblast function, including Wnt/β-catenin signaling. Of the 22 most significantly regulated (ANOVA p<0.01) miRNAs between non-arthritic and arthritic mice, 11 were predicted to target numerous Wnt and BMP signaling pathway components. We validated their expression in isolated synovial fibroblasts and in whole synovium by qPCR and showed that 4 of these miRNAs were downregulated by TNF. Some of the target genes of these miRNAs, including GSK3β, Sfrp2 and Tob1, were up-regulated at erosion sites in bone. Among the 11 miRNAs, miR-221 was significantly up-regulated during peak inflammation. Since miR-221 is known to be up-regulated in RA synovial tissues (Pandis et al., 2012), we examined the role of miR-221 in osteoblasts. Transfection of miR-221 into primary murine calvarial osteoblasts suppressed osteoblast differentiation and mineralization. Expression of a predicted target gene of miR-221 that is essential for bone mineralization, DKK2, is suppressed by miR-221 at the protein level. Tcf-1, a downstream target of Wnt signaling, is also down-regulated by miR-221. 

Conclusion: We have identified several miRNAs expressed in inflamed synovium that potentially regulate skeletal pathways, including osteoblast differentiation. Of these, miR-221 inhibits osteoblast differentiation. These results support the hypothesis that miRNAs derived from inflamed synovial tissue may regulate skeletal pathways and osteoblast function in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/synovium-derived-micrornas-inhibit-bone-formation-in-rheumatoid-arthritis/