Method: RA FLS were isolated from synovial tissues from different patients. Cells were stimulated with TLR2 (BLP) and TLR4 (LPS) ligands and quantitative RT-PCR was performed to evaluate miRNA and mRNA expression in RA FLS. Transient transfection of FLS with mimic 15a 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-15a mimic or antagomir was performed using Lipofectamine. Luciferase activity were determined using the dual-luciferase reporter assay system. IL-6 release was measured in culture supernatants by ELISA according to the manufacturer’s instructions.
Result: We first showed that RA FLS expressed constitutively Etk, and that its expression is up-regulated in response to LPS and BLP. To identify miRNA targeting Etk mRNA, RA FLS were activated with LPS for six hours and a miRNA microarray analysis was performed. Among the down-regulated miRNAs, miR-15a was predicted to target Etk. This down regulation was confirmed by quantitative RT-PCR. As in silico analysis predicted that miR-15a might possibly bind to the 3’-UTR of human Etk transcript, we transiently transfected into HEK-293 cells a reporter construct that contain the firefly luciferase gene fused to the Etk 3’-UTR containing the putative miR-15a interactor site along with miR-15a. We observed a downregulation of the luciferase activity, indicating that the 3’-UTR of Etk mRNA is directly targeted by miR-15a. Moreover transfection of RA FLS using miR-15a mimics decreased IL-6 release in response to LPS and BLP.
Conclusion: These results suggest an important role of miR-15a in the control of Etk expression and IL-6 synthesis and indicate that its expression may be critical to prevent an excessive inflammatory response.
Disclosure: G. Alsaleh, None; L. Philippe, None; A. Pichot, None; S. Pfeffer, None; J. E. Gottenberg, None; J. Sibilia, None; P. Georgel, None; D. Wachsmann, None.