Abstract
IntroductionAbnormal toll-like receptor (TLR)3 signaling plays an indispensable role in pathogenesis of both experimental and human rheumatoid arthritis, and microRNAs (miRNAs) might orchestrate this signaling pathway. This study was performed to determine the relationship between miR-26a and TLR3 in rat macrophages and to observe effects of miR-26a mimic on pristane induced arthritis (PIA) in rats.MethodsDual luciferase reporter assay was used to validate the direct interaction between miR-26a (a candidate miRNA to target tlr3 mRNA) and tlr3 3′UTR. MiR-26a regulation on TLR3 gene expression was determined using RT-qPCR and Western blotting after miR-26a mimics and inhibitors were transfected into rat macrophage line NR8383 cells. Poly I:C (TLR3 ligand) was used to trigger TLR3 activation, and mRNA expression of its downstream cytokines interferon (ifn)-β and tumor necrosis factor (tnf)-α was accordingly detected to determine the regulation of TLR3 signaling. Expressions of TLR3 and miR-26a were detected during rat bone marrow derived macrophage (BMDM) induction, in pristane stimulated NR8383 cells and spleens from methotrexate (MTX) treated PIA rats. A miR-26a mimic was administrated intraperitoneally to PIA rats, and arthritis severity was evaluated by macroscopic or microscopic observations.ResultsDirect target relationship between miR-26a and tlr3 mRNA in rats was confirmed. Modifications of miR-26a function by transfection of miR-26a mimics and inhibitors exhibited corresponding repression and augmentation of TLR3 and its signaling downstream cytokine expressions in NR8383 cells. The alteration of miR-26a expression was negatively related with TLR3 expression during BMDM induction, in pristane-primed NR8383 cells and PIA rat spleens. Moreover, both abnormal expressions were rescued in MTX treated arthritis rat spleens. The miR-26a mimic treatment displayed the depression of TLR3 expression and ameliorated the disease severity in the rats with pristane induced arthritis.ConclusionsMiR-26a negatively regulates TLR3 signaling via targeting of TLR3 itself in rat macrophages, and this finding provides a novel insight into abnormal TLR3 overexpression during experimental arthritis.
Highlights
Abnormal toll-like receptor (TLR)3 signaling plays an indispensable role in pathogenesis of both experimental and human rheumatoid arthritis, and microRNAs might orchestrate this signaling pathway
The activated TLR3 pathway could further promote rheumatoid arthritis synovial fibroblast (RASF) sustaining B cell activation in the synovium [9]. We found that both TLR3 mRNA and protein expressions are prominently upregulated in splenic macrophages in rats with pristine-induced arthritis (PIA) and collageninduced arthritis (CIA), and downregulation of TLR3 expression modulates the severity of arthritis [10,11]
As it could bind to tlr3 mRNA from diverse species, including bushbabies, mice, rabbits and armadillos, miR-26a was chosen for further investigation
Summary
Abnormal toll-like receptor (TLR) signaling plays an indispensable role in pathogenesis of both experimental and human rheumatoid arthritis, and microRNAs (miRNAs) might orchestrate this signaling pathway. Recent studies have demonstrated that TLR3 is involved in the pathogenesis of virus infection and autoimmune disorders, especially RA, in which RA synovial fibroblasts (RASFs) from early-stage patients highly express TLR3 and react with its ligand in vitro, suggesting that this pathway is activated early in the disease process [6,7]. RASFs are activated by stimulation with both synthetic and endogenous TLR3 ligands such as poly I:C and necrotic RA synovial fluid cells, resulting in pro-inflammatory gene expression [8]. The findings indicate that excess and persistent expression of the TLR3 gene in macrophages and synovial cells could be responsible for arthritis development
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