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I.A. de la Rosa et al.
Figure 2. Bioinformatic identification of deregulated miRNA and protein targets related to the pathogenic profile of neutrophils in rheumatoid arthritis. In silico studies were performed to identify eight altered miRNA using QIAGEN’s Ingeunity Pathway Analysis (IPA, QIAGEN Redwood City, USA; https://analysis.ingenuity.com) as the main regulators of proteins involved in the abnormal activation of neutrophils in RA: inflammation, migration and cell survival. By using the tool miRNA target filter of IPA, a network including the selected miRNA and mRNA targets experimentally observed and predicted with high confidence, was generated. miRNA: microRNA; RA: rheumatoid arthritis.
In vitro treatment of active neutrophils purified from RA patients with IFX restored the low levels of the eight selected miRNA while TCZ only up-regulated the expres- sion of miR-148a (Figure 5D). Accordingly, IFX upregulat- ed the expression levels of AGO1. Regarding mRNA tar- gets, IFX reduced the mRNA expression of VEGF-A, TNF-a, IL-1β, IL-8 and STAT3. Treatment with TCZ also diminished the expression of various of these mRNA tar- gets such as IL-1β, IL-8, IL-6R and STAT3 (Figure 5E). In addition, protein release of TNF-a, IL-8 and IL-1β was reversed in RA neutrophils after treatments with both, IFX and TCZ (Figure 5F).
Overexpression of miR-126, miR-148a and miR-223 in RA neutrophils decreases specific targets involved in inflammation, migration and cell survival
We selected three downregulated miRNA to evaluate their role in migration, proinflammatory profile and cell survival of RA neutrophils: miR-223 was the most abun- dant in neutrophils, miR-148a and miR-126 have several potential and demonstrated mRNA targets involved in inflammation. As visible in Figure 6, miR-126, miR-148a and miR-223 overexpression led to a downregulation of their specific mRNA targets: miR-126 overexpression induced a significant downregulation of VEGF-A protein expression (Figure 6A), and miR-223 overexpression pro- moted a significant decrease in the protein expression of IL-8 and IL-1 . Interestingly, miR-223 transfection induced
a significant increase of VEGF mRNA and protein (Figure 6C). On the other hand, miR-148a overexpression reduced gene and protein expression levels of TNF-a (Figure 6B).
DICER downregulation in neutrophils might exacerbate their inflammatory profile
Using a low number of lentiviral particles, 25% of DICER expression was inhibited in HL-60 neutrophil-like cells (Figure 6D). This reduction promoted a significant decrease in all the selected miRNA (Figure 6E). Protein lev- els of a number of cytokines and chemokines were signif- icantly upregulated in neutrophils after DICER downregu- lation (Figure 6F).
Discussion
This study reports for the first time the altered miRNA expression profile in neutrophils from RA patients, describing a defect in the miRNA processing machinery responsible for a global low abundance of miRNA, medi- ated by ACPA and inflammatory mediators, promoting the high inflammatory profile of these cells in RA.
Several miRNA have been shown to be increased in the PB and in inflamed joints in RA patients, correlated to dis- ease activity and to promoting the production of inflam- matory mediators involved in the synovitis.13,14 Likewise,
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