Article Text

THU0039 Regulation of ASK-1 Expression by Microrna-17 and Its Correlation with Rheumatoid Arthritis Pathogenesis
  1. N. Akhtar,
  2. S. Ahmed
  1. Pharmaceutical Sciences, Washington State University, Spokane, United States


Background Deregulated expression of microRNAs (miRs) has shown to be critical for the pathogenesis of rheumatoid arthritis (RA)1,2. Apoptosis signal regulating kinase (ASK)-1, a member of MAP3K family upstream of p38 MAPK has recently been associated with RA pathogenesis3.

Objectives To evaluate the role of ASK-1 in RA pathogenesis and its epigenetic regulation by miRNA in human RA synovial fibroblasts (RA-FLS) and rat adjuvant-induced arthritis (AIA) model.

Methods Bioinformatics analysis was performed to identify the predicted miRNA targets at 3'UTR region of ASK-1 gene. ASK-1 expression in FLS from RA, osteoarthritis (OA), or non-diseased (NH) donors was compared and correlated with miR-17 expression using qRT-PCR and Western immunoblotting. Effect of IL-1β (10 ng/ml) or TNF-α (20 ng/ml) stimulation alone or in the presence of ASK-1 inhibitor (TC-ASK-10) on ASK-1 mRNA and protein and IL-6 production was examined using ELISA. Transfection of RA-FLS with a 3'UTR reporter construct and pre-miRNAs was performed to verify the miRNA: mRNA interaction. Effect of overexpression and inhibition of miR-17 on ASK-1 expression was evaluated by transient transfections of RA-FLS. Findings from human FLS were validated in vivo using a rat AIA model.

Results Bioinformatics analysis predicted miR-17 binding sites at 3'UTR region of ASK-1. MiR-17 expression was down-regulated by ∼70% in RA-FLS as compared to NH-FLS (p<0.05; n=5) and inversely correlated with a significant increase in the expression of ASK-1 mRNA (∼2.3-fold) and protein (p<0.05). However, no significant correlation was observed between miR-17 or ASK-1 expression in OA-FLS compared to NH-FLS or tissue. Stimulation of RA-FLS with IL-1β or TNF-α for 24h further suppressed the expression of miR-17, with a concomitant increase in ASK-1 mRNA (∼2.2 to 12.5 fold) and protein expression (p<0.05). Interestingly, inhibition of ASK-1 using TC-ASK-10 (12.5 μM) resulted in a marked attenuation of p-p38 MAPK and IL-6 expression in RA-FLS. Co-transfection of RA-FLS with the ASK-1 luciferase reporter construct and pre-miR-17 significantly suppressed the luciferase activity by ∼29% (p<0.05). Overexpression of miR-17 inhibited the expression of ASK-1 (∼25%), whereas transfection with miR-17 inhibitors enhanced its expression (∼22%) in RA-FLS (p<0.05). In rat AIA model, ASK-1 expression increased in with the disease progression, and correlated with the reduced miR-17 expression and enhanced p-p38 MAPK and IL-6 expression. These data suggest that ASK-1 mRNA is a direct target of miRNA-17.

Conclusions This study provides evidence for the role of ASK-1 in RA pathogenesis and a novel mechanism of its epigenetic regulation by miR-17 in RA-FLS and rat AIA model.


  1. Wittman J, et al. Ann. Rheum. Dis. 2011; 70 (Suppl 1): i92-i96.

  2. Duroux-Richerd I, et al. Arthritis Rheum. 2012; 64(1):11-20.

  3. Mnich SJ, et al. Int. Immunol. 2010;10:1170-76.

Acknowledgements This study was supported by the NIH grant AR063104 (S.A.), The Arthritis Foundation Innovative Research Grant (S.A.), and the Start-up funds from Washington State University.

Disclosure of Interest None declared

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