THU0025 MICRO-RNA DIFFERENTIALLY REGULATE THE ALTERNATIVE PRTN3-MRNA IN GRANULOMATOSIS WITH POLYANGIITIS
Authors: N. Reichard, A. Kerstein-Staehle, A. Müller, G. Riemekasten, P. Lamprecht, S. Schinke
Journal: Annals of the Rheumatic Diseases (2020)

Background: Micro-RNAs (miRNA) are short non-coding RNAs that regulate inflammation mostly by translational repression. Previously, we screened 847 miRNAs in nasal tissue from GPA patients and found a disease associated alteration of miRNA expression compared to healthy controls and chronic rhinosinusitis. MiR-184 was most over expressed in nasal tissue from GPA (13.4x). The dual-luciferase reporter assay confirmed a significant reduction of Proteinase-3 (PRTN3) expression by miR-184 (1). PRTN3 transcripts with an alternative 3’ untranslated region (UTR) have been described in GPA (2). The pathophysiological relevance of this alternative transcript remains unclarified. Objectives: To identify new miRNA targets of potential pathophysiological relevance in GPA, we validated the effect of the 21 most dysregulated miRNAs on the mRNA of PRTN3. Further, we included the alternative PRTN3 mRNA in our screen to look for new regulatory differences. Methods: The inhibitory capacity of miRNAs on Proteinase-3 mRNA was estimated by a dual-luciferase reporter system. The sequences of the alternative (132bp longer) and the regular 3’UTR-PRTN3 were cloned and inserted into the pmirGLO vector and co-transfected with 21 miRNA mimics into HeLa cells. Co-transfection with Caenorhabditis elegans miRNA 67 mimic (cel-miR-67) was used as negative control. Statistical significance was evaluated by students t-test adjusted for multiple comparisons (Holm-Sidak). Results: For 18 of 21 investigated miRNAs no effects could be observed on the alternative and the regular 3’UTR-PRTN3. But there were remarkable differential effects of let-7f, miR-184 and miR-708. Let-7f (-29,2%) and miR-708 (-23,6%) both showed a suppression of the alternative 3’UTR-PRTN3 but no effect on the regular 3’UTR-PRTN3 while miR-184 only suppressed the regular 3’UTR (-17,5 %) and not the alternative variant (fig. 1-2). Fig. 1. Dual-luciferase reporter assay with the regular 3’UTR of PRTN3 cloned into the pmirGLO vector compared to empty vector (NTC). Significant effect for miR-184 (17,5 %), miR-708 no effect and let-7f small but not significant reduction in luciferase activity (12,3 %). Data represent 3 independent experiments with triplicate measurements. miR-184 was tested 6 times. *P&lt;0.05; ns = not significant; error bars display standard deviation. Fig. 2. Dual-luciferase reporter assay with the alternative 3’UTR of PRTN3. Significant effects of let-7f (29,2 %) and miR-708 mimic (23,6 %) but no significant effects of miR-184 of luciferase activity. 3 independent experiments with triplicate measurements. *P&lt;0.05 Conclusion: Disease specific miRNA signatures together with an increased PRTN3 level and in alternative PRTN3 mRNA in GPA suggest a dysregulation of PRTN3 expression in GPA. To our knowledge this is the first analysis in GPA showing that miRNAs can differentially regulate the expected and the alternative 3’UTR variants of PRTN3-mRNA. As miR-184 is markedly upregulated in GPA, a repression of PRTN3 is to be anticipated, possibly as a reaction to previous neutrophil activation with PRTN3 overexpression. Our findings also strengthen the potential pathophysiological role of the alternative PRTN3 mRNA. References: [1] Schinke S et al PROTEINASE-3 REGULATING MICRO-RNA IN GRANULOMATOSIS WITH POLYANGIITIS. Ann Rheum Dis 2019 (78 Suppl 2):437 [2] McInnes E et al Dysregulation of Autoantigen Genes in ANCA-Associated Vasculitis Involves Alternative Transcripts and New Protein Synthesis J Am Soc Nephrol. 2015 26(2): 390–399 Acknowledgments: Vasculitis foundation for funding Disclosure of Interests: Nick Reichard: None declared, Anja Kerstein-Staehle: None declared, Antje Müller: None declared, Gabriela Riemekasten Consultant of: Cell Trend GmbH, Janssen, Actelion, Boehringer Ingelheim, Speakers bureau: Actelion, Novartis, Janssen, Roche, GlaxoSmithKline, Boehringer Ingelheim, Pfizer, Peter Lamprecht: None declared, Susanne Schinke Speakers bureau: Pfizer