In rheumatoid arthritis (RA), the fibroblast-like synoviocytes (FLS), resident cells of the joint space, show an aggressive phenotype. They release a broad range of cytokines and enzymes which contribute to destruction. Extrinsic factors such as microbial components (PAMPs) or damage associated molecular patterns (DAMPs) activate FLS by interacting with Pattern Recognition Receptors (PRRs) such as Toll like receptors (TLRs). But it becomes more and more evident that epigenetic factors such as miRNAs play an important regulatory role in the development of the inflammatory response in RA. Our group identified in LPS-activated RA FLS a miRNA, miR-346 which functioned as a negative regulator of IL-18 and TNF-α release by activated FLS. We also demonstrated that miR-19 (miR-17-92 cluster) controlled directly TLR2 expression in RA FLS. An online search of the miRBase target database, demonstrated that one miRNA: miR-20a belonging to the downregulated miR 17-92 cluster was predicted to potentially target the 3’-UTR region of a mitogen-activated protein 3-kinase (ASK1) selectively required for LPS-induced activation. In this study, we aimed to evaluate the role of miR-20a in the regulation of this TLR signalling component.
RA FLS were isolated from synovial tissues and stimulated with TLR2 (BLP) and TLR4 (LPS) ligands. QRT-PCR was performed to evaluate miRNA and mRNA expression. Transient transfection of FLS with mimic 20a was performed using the Human Dermal Fibroblast NucleofectorTM kit from Amaxa. All assays were performed 48 h post transfection. Transfection of HEK293 cells with reporter constructs and miR-20a mimic or antagomir was performed using Lipofectamine. Luciferase activity was determined using the dual-luciferase reporter assay system. IL-6 release was measured in culture supernatants by ELISA.
We first showed by qRT-PCR that RA FLS expressed constitutively ASK1 and that ASK1 expression is significantly enhanced in response to LPS and BLP. Consistent with the downregulation of miR-17-92 cluster expression, miR-20a was strongly down regulated in RA FLS activated by LPS and BLP. As in silico analysis predicted that miR-20a might possibly bind to the 3’-UTR of human ASK1 transcript, we transiently transfected into HEK-293 cells, a reporter construct that contain the firefly luciferase gene fused to the ASK1 3’-UTR containing the putative miR-20a interactor site along with miR-20a. We observed a downregulation of the luciferase activity, indicating that the 3’-UTR of ASK1 mRNA is directly targeted by miR-20a. Finally, to examine the consequence of a decrease of ASK1 expression by miR-20a, we tested by transfection, whether expression of miR-20a affected IL-6 release in LPS-activated RA FLS. As compared to the control, we observed that transfection of the mimics induced a strong downregulation of IL-6 release in response to LPS and not to BLP.
These results illustrate a negative feedback loop that control TLR4 response. Our data strongly suggest a critical role of miR-20a in the regulation of the expression of ASK1 in response to TLR4 activation which could play an important role in the regulation of the inflammatory response in RA.
Disclosure: L. Philippe, None; G. Alsaleh, None; S. Pfeffer, None; J. E. Gottenberg, None; J. Sibilia, None; D. Wachsmann, None; P. Georgel, None.