POS0998 SALIVARY GLANDS FROM PATIENTS WITH SJÖGREN’S SYNDROME SHOW ALTERED EXPRESSION OF COMPONENTS OF THE MICRORNA BIOGENESIS MACHINERY THAT CORRELATE WITH INFLAMMATORY PARAMETERS
Authors: S. Aguilera, P. Carvajal, I. Castro, M. J. Barrera, S. González, C. Molina, M. J. González
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> MicroRNAs (miRNAs) are small non-coding RNAs involved in the regulation of gene expression at the post-transcriptional level (by translational blocking or mRNA degradation). The canonical pathway of miRNA biogenesis involves the transcription of the primary miRNA and its processing by the multiprocessor complex (consisting of the RNAse Drosha and its cofactor DGCR8), generating the miRNA precursor (pre-miRNA). The pre-miRNAs are transported to the cytosol where the RNAse Dicer processes them. TRBP and PACT (two Dicer binding partners), promote Dicer positioning at the 3' end of the pre-miRNA for processing to a double strand miRNA. The mature miRNA strand is transferred to Ago proteins forming the RNA-induced silencing complex (RISC). Stressor stimuli, such as inflammatory mediators, modify the levels, localization and/or activity of key miRNA-bioprocessing core components. Previously, in labial salivary glands (LSG) of Sjögren's syndrome (SS) patients, we found altered levels of miRNAs involved in the regulation of genes essential for glandular homeostasis and secretory function [1]. However, it is not yet known whether the alterations in the expression pattern of miRNAs in LSG from SS patients are due to changes in their metabolic machinery induced by inflammation. <h3>Objectives:</h3> To characterize the expression and localization of microRNA biogenesis components in LSG from primary SS patients and to evaluated if proinflammatory cytokines modify their levels or distribution in vitro. <h3>Methods:</h3> LSGs biopsies from 12 primary SS patients and 11 non-Sjögren sicca controls were studied. Human salivary glands (HSG) cells stimulated with 10 ng/mL of TNF-α, IFN-α, IFN-β or IFN-γ for 24 h were also evaluated. The mRNA and protein levels, as well as localization of Drosha, DGCR8, Dicer, TRBP, PACT, Ago1, Ago2 and Ago4 were determined by qPCR, Western blot, and immunofluorescence. Spearman's rank correlation analyses were performed between mRNA/protein levels and clinical parameters. <h3>Results:</h3> Our results showed reduced mRNA levels of Drosha, Dicer, PACT, TRBP and Ago1 in LSGs from SS patients, while protein levels showed great variability among SS patients. Decreased mRNA levels of Drosha, Dicer and TRBP were inversely correlated with focus score and EULAR SS disease activity index (ESSDAI). Drosha, DGCR8, DICER, PACT and Ago2 were detected in the cytoplasm, and TRBP was unexpectedly observed in the nuclei. In HSG cells stimulated with proinflammatory cytokines (TNF-α, IFN-α, IFN-β or IFN-γ), increased mRNA and protein levels of Drosha, DGCR8, Dicer, PACT, TRBP, Ago2 and Ago4 were observed. Dicer staining was weaker in cells stimulated with IFN-β, IFN-γ and TNF-α, while TRBP signal was weaker in cells stimulated with IFN-β and IFN-γ. <h3>Conclusion:</h3> Altered expression of miRNA biogenesis machinery components correlated with focus score and ESSDAI, together with alterations induced by pro-inflammatory cytokines in vitro, contribute to understand the mechanisms underlying the differential expression of miRNAs in LSGs from SS patients. Future studies using anti-inflammatory therapies may restore miRNA metabolism improving glandular function in SS patients. <h3>REFERENCES:</h3> [1] Castro et al, 2022. Small RNA Expression Profiling Reveals hsa-miR-181d-5p Downregulation Associated With TNF-α Overexpression in Sjögren's Syndrome Patients. Front Immunol 13:870094. doi: 10.3389/fimmu.2022.870094. eCollection 2022. <h3>Acknowledgements:</h3> This study was supported by Fondecyt ANID 1210055, Fondecyt ANID 1230852, Fondecyt Iniciación 11201058, Fondecyt Postdoctorado 3240081. <h3>Disclosure of Interests:</h3> <b>None declared.</b>