Background/Purpose: Aberrant expression of microRNAs (miRs) has shown to be critical for the pathogenesis of rheumatoid arthritis (RA). Apoptosis signal regulating kinase-1 (ASK1), a member of MAP3K family upstream of p38 and JNK MAPK has recently been associated with RA pathogenesis. Here we studied the regulation of ASK1 by miR-17 in human RA synovial fibroblasts (RA-FLS) and correlated their expressions in human RA and adjuvant induced arthritis (AIA) and collagen induced arthritis (CIA) models of RA.

Methods: Bioinformatics analysis was performed to identify miRs predicted to targets ASK1 3’UTR. ASK1 and miR-17 expressions were correlated in FLS/synovial tissue (ST) from RA (n=15), osteoarthritis (OA; n=11), or non-diseased (NL; n=8) donors using Western blotting and qRT-PCR. Effect of IL-1β (10 ng/ml) or TNF-α (20 ng/ml) with or without ASK1 inhibitor (TC-ASK-10; 12.5 μM) or ASK1 siRNA was studied on the expression of phospho-p38 (p-p38), p-JNK, IL-6, and IL-8. Luciferase assay was performed using ASK1 3’UTR construct and pre-miR-17 to verify miRNA:mRNA interaction. Effect of miR-17 overexpression and inhibition on ASK1 was evaluated by transfection of RA-FLS with pre-miR-17/anti-miR-17. Findings from human FLS were validated in AIA and CIA models. Serum miR-17 levels in RA patients, AIA and in CIA models were quantified using qRT-PCR. Effect of miR-17 overexpression on matrix metalloproteinases (MMPs) was determined in the conditioned media using MMPs protein array. p<0.05 was considered significant.

Results: Bioinformatics analysis predict miR-17 binding site in the 3’UTR region of ASK1 mRNA. Expression of miR-17 was down-regulated (70%) in RA-FLS compared to NL-FLS and inversely correlated with a significant increase in ASK1 mRNA (2.3-fold) and protein. Similar inverse correlation of miR-17 and ASK1 was also observed in RA-ST compared to NL-ST. No significant correlation was observed between miR-17 and ASK1 expression in OA-FLS or OA-ST. IL-1β or TNF-α stimulation further suppressed miR-17 expression, with a concomitant increase in ASK1 mRNA (2.2-12.5 fold) and protein in RA-FLS. Co-transfection of RA-FLS with the ASK1 reporter construct and pre-miR-17 significantly suppressed the luciferase activity (34%). Overexpression of miR-17 inhibited ASK1 protein, whereas its inhibition enhanced ASK1 protein in RA-FLS. Interestingly, inhibition of ASK1 using TC-ASK-10 resulted in a marked attenuation of p-p38, p-JNK MAPK, IL-6, and IL-8 expression in RA-FLS. Knockdown of ASK1 or overexpression of miR-17 inhibited TNF-α-induced IL-6 and IL-8 production in RA-FLS. Overexpression of miR-17 also inhibited MMP-1, -3, and -13 production in IL-1β stimulated RA-FLS. miR-17 levels were significantly lower in the serum of RA patients, AIA and in CIA models compared to controls with increased in arthritis score. In AIA and CIA studies, ASK1 expression increased with the disease progression, and correlated with lower miR-17 expression and enhanced p-p38, p-JNK, IL-6, and IL-8 expression in arthritic joints.

Conclusion: This study provides evidence for the role of ASK1 in RA pathogenesis and a novel mechanism of its regulation by miR-17 in human RA-FLS and in two different models of RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/regulation-of-ask1-expression-by-microrna-17-and-their-correlation-with-rheumatoid-arthritis-pathogenesis/