THU0316 PROTEINASE-3 REGULATING MICRO-RNA IN GRANULOMATOSIS WITH POLYANGIITIS
Authors: Susanne Schinke, Nick Reichard, Barbara Russo, Antje Müller, Martin Laudien, Robert Häsler, Gabriela Riemekasten, Peter Lamprecht
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> Dysregulated miRNA expression profiles have been described in diverse chronic inflammatory diseases. We previously did a microarray screening of 847 miRNAs in nasal tissue from GPA patients and we found a disease associated alteration of miRNA expression compared to healthy controls (HC) and chronic rhinosinusitis (CRS). <h3>Objectives:</h3> In order to identify new miRNA targets of potential pathophysiological relevance in GPA, we validated dysregulated miRNAs by qPCR in GPA nasal tissue biopsies and sera. Moreover, we screened GPA associated miRNAs for their potential to regulate proteinase-3 (PRTN3). <h3>Methods:</h3> In an independent validation cohort (tissue and sera from 14 GPA-patients, 10 disease controls: CRS and Crohn's/CD) 12 miRNAs were examined by qPCR. Validated and computational miRNA targets were identified by mirDIP algorithms. The inhibitory capacity of miRNAs on Proteinase-3 (PRTN3) was estimated by a dual-luciferase reporter system (Promega®). The 3'UTR-PRTN3 sequence was cloned and inserted into the pmirGlo vector and co-transfected with the hsa-mirna mimics (Dharmacon®) into HeLa cells. As a second method, the effect of miR-184 transfection on the endogenous PRTN3 expression in the human myeloid leukemia cell line HL-60 was estimated by western blot. <h3>Results:</h3> Microarray screening revealed alterations of 24 miRNAs in GPA nasal tissue compared to HC and CRS. qPCR confirmed dysregulation of 6 tissue related miRNAs also in GPA sera. Compared to CD 4 miRNAs (miR-10b, -99a/100, -125b, -532–3p) were down regulated in GPA tissue. The miRNA with the highest expression level in nasal tissue from GPA was miR-184. miR-184 along with miR-708 and mir-214-5p were also predicted to target PRTN3 by the mirDIP algorithm. The dual-luciferase reporter assay revealed a significant reduction of PRTN3 expression by miR-184, while these effects could not be observed for miR-708 or miR-214-5p. The transfection of miR-184 into HL-60 cells resulted in a dose-dependent knockdown of PRTN3 expression as detected by Western blot. <h3>Conclusion:</h3> Characteristic miRNA signatures in GPA, CRS and CD suggest distinct pathophysiological mechanisms. It indicates at a local miRNA dysregulation in inflamed GPA tissue with a corresponding serum signature that might serve as novel biomarkers. To our knowledge this is the first analysis that attempts to correlate GPA-associated miRNA expression patterns in tissue with serum. Moreover, this is the first description of a miRNA (miR-184) that potentially regulates the expression of the GPA autoantigen PRTN3. <h3>References:</h3> [1] O'Connell R et.al. Physiological and pathological roles for microRNAs in the immune system. Nat Rev Immunol. 2010;10(2):111-122. [2] Fasseu M et al. Identification of restricted subsets of mature microRNA abnormally expressed in inactive colonic mucosa of patients with inflammatory bowel disease. PLoS One. 2010 Oct 5;5(10). [3] Neudecker, V. MicroRNAs in mucosal inflammation. J Mol Med (Berl). 2017 September; 95(9): 935–949 [4] Coit P et al. An update on the role of epigenetics in systemic vasculitis. Curr Opin Rheumatol. 2018; 30(1): 4–15. <h3>Disclosure of Interests:</h3> Susanne Schinke Grant/research support from: travel and congress expenses from different companies pfizer, ucb, chemocentryx, Janssen-Cilag, msd, Nick Reichard: None declared, Barbara Russo: None declared, Antje Müller: None declared, Martin Laudien Paid instructor for: Olympus, Speakers bureau: Novartis, Robert Häsler: None declared, Gabriela Riemekasten Consultant for: Chugai, F. Hoffmann-La Roche, Speakers bureau: Chugai, F. Hoffmann-La Roche, Peter Lamprecht: None declared