82. Differential effects of miR-184, overexpressed in GPA, on PRTN3 and AKT2 in a granulocyte model
Creators: name: Susanne Schinke1, 2
name: Hinsch1, Maximilian
name: Reichard1, Nick
name: Pommerien1, Lena
name: Judycka1, Anna
name: Müller1, Antje
name: Peter Lamprecht1, 2
name: Kerstein-Stähle1, Anja
Publisher: name: Zenodo
Year: 2022
Subjects: Vasculitis
ANCA
Abstracts
Giant Cell Arthritis
MPA
GPA
EGPA
Takayasu
IgA vasculitis

description: <strong>Background</strong>: MicroRNAs (miRNAs) regulate a wide range of biologic and pathologic processes including inflammation mainly through translational repression. Previously, we screened nasal tissue from granulomatosis with polyangiitis (GPA) patients for 847 miRNAs and found a disease associated signature of 21 differentially expressed miRNAs compared to healthy controls and chronic rhinosinusitis. MiR-184 was most over expressed in nasal tissue from GPA (13.4fold) and it is predicted to target the proteinase-3 (PR3) gene PRTN3. First, we aimed to confirm if any of 21 GPA-associated miRNAs including miR-184 downregulates PRTN3 or its alternative transcript in vitro. The second objective was to validate miR-184 as a putative regulator of PR3 on mRNA and protein level in a granulocyte model. <strong>Methods:</strong> The inhibitory capacity of miRNAs on PR3 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 <em>Caenorhabditis elegans </em>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). NB4 cells were differentiated by all-transretinoic acid for 24h. Granulocyte-like differentiation was analyzed by FACS, qPCR and western blot. Endogenous miR-184 expression was examined by miRNA-specific PCR. miR-184 and the GPADH miRNA control were transfected using lipofection. Downregulation of target genes and proteins were analyzed by qPCR after 24h and by western blot after 48h. <strong>Results:</strong> From the selected 21 miRNAs 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).<br> NB4 cells were differentiated into a CD11b+/CD14- granulocyte-like phenotype expressing PR3 mRNA and protein. Endogenous miR-184 expression was not found. Downregulation of GAPDH by control miRNA was validated on protein level. We could not confirm that miR-184 regulates PR3 on mRNA or protein level down, but we could show a downregulation of AKT2 on mRNA and protein level by miR-184. <strong>Conclusions</strong>: Disease specific miRNA signatures together with an increased PRTN3 level and in alternative PRTN3 mRNA in GPA suggest an epigenetic dysregulation of PRTN3 expression in GPA. To our knowledge this is the first analysis in GPA showing that miRNAs can differentially regulate the regular 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. However, we could not confirm downregulation of PR3 on mRNA or protein level in the NB4 granulocyte model. But AKT2, an alternative validated target of miR-184, was downregulated serving as positive control of our cell model. Interestingly, AKT2 is involved in cell death mechanism such as apoptosis. Since miR-184 is highly overexpressed in nasal tissue of GPA patients, the miR-184/AKT2-axis might represent a potential new link to mechanisof cell death regulation, driving autoimmunity in GPA. <strong>Disclosures: </strong>S. Schinke: grant by vasculitis foundation Kansas City, MO 64188. Other authors: none <br>