Objective To evaluate whether miR-20a belonging to the cluster miR-17–92 is a negative regulator of inflammation in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) by modulating expression of apoptosis signal-regulating kinase (ASK) 1, a key component of the toll-like receptors 4 pathway, upstream of p38 mitogen-activated protein kinase.
Methods Evaluation of miR-20a and ASK1 mRNA was performed by RT-qPCR. ASK1 protein expression was assessed by western blotting. Overexpression of miR-20a was performed by transfection of RA FLS and THP-1 cells with miR-20a mimics. Interleukin (IL)-6, CXCL-10, IL-1β and TNF-α release were measured by ELISA. The role of miR-20a in vivo was assessed by IL-6 release from macrophages obtained from mice injected intraperitoneally with vectorised miR-20a mimics.
Results We showed that stimulation of RA FLS with lipopolysacharide (LPS) and bacterial lipoproteins (BLP) induces a drop in expression of miR-20a and that this decrease is associated with an upregulation of ASK1 expression. Using transfection of Ask1 3′UTR reporters, we demonstrate that Ask1 is a direct target of miR-20a. Overexpression of miR-20a led to a global decrease in ASK1 protein in BLP- and LPS-activated cells indicating that miR-20a regulates the expression of ASK1 at the translational level. Transfection of miR-20a mimics decreases IL-6 and CXCL10 release by RA FLS and IL-1β and TNF-α by activated THP-1 cells but only in response to LPS. Last, injection of vectorised miR-20a mimics to mice led to a global decrease in ASK1 protein expression and IL-6 secretion in LPS-activated macrophages.
Conclusions Our data point toward an important role for miR-20a in the regulation of pro-inflammatory cytokines release, by controlling ASK1 expression in RA FLS.
- Rheumatoid Arthritis
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Correction notice This article has been updated since it was published Online First. An authorship statement has been added.
Contributors LP, GA, AP and EO performed the experiments and participated in data analysis. GZ and BF contributed reagents. JS, SP, PG and DW designed the experiments, analysed the data and wrote the manuscript. SB participated in data analysis.
Funding INSERM and Université de Strasbourg supported this work. Pr. Wachsmann acknowledges financial support from Bristol Myers Squibb, Roche, Pfizer and CAMPLP. Work in the laboratories of PG and SP is funded by Agence Nationale de la Recherche (ANR) (ANR- 08-MIEN-005-01 DICERPATHO). Work in the laboratory of SP is also supported by the European Research Council (ERC Starting Grant ncRNAVIR 260767).
Competing interests None.
Ethics approval Informed consent was provided according to the Declaration of Helsinki and obtained from all patients. Approval by the ethical committee of the Hopitaux Universitaires de Strasbourg was obtained.
Provenance and peer review Not commissioned; externally peer reviewed.