P132 Overexpression of miR-376a-3p in exosomes circulating in peripheral blood of patients with Crohn's disease

Abstract

Abstract Background MicroRNAs (miRs) can modulate the development and/or progression of chronic inflammation in patients with Crohn's disease (CD), influencing the efficacy of treatments. Aim The objective was to assess the miRs content of circulating exosomes in the peripheral blood of patients with CD to identify new biomarkers useful in disease management. Methods Patients with CD diagnosed and followed up at the Hospital General Universitario de Alicante and healthy controls were included. Extracellular vesicles were isolated from serum and exosomes were quantified by "Nanoparticle Tracking Analysis" (NTA), confirmed by Transmission Electron Microscopy (TEM) and by specific markers (Tsg101, CD63, CD81) by Western blot, performing an expression analysis of a non-targeted panel of human miRs from exosomal RNA. Patient and control samples were also subjected to enzyme-linked immunosorbent assay (ELISA) measurement of different target-predicted protein substrates. Results Thirty patients and ten controls were included in the study. Patients were 42±9 years old, disease duration 62±9 months, 40% women and 80% showed ileal or ileocolonic involvement. Six patients (20%) had perianal disease. Ninety-three per cent were undergoing biological treatment with infliximab (IFX, n=13), adalimumab (ADA, n=8) or ustekinumab (USTE, n=7). A significant increase in exosome concentration was observed in the serum of patients vs controls. NTA and TEM confirmed the size and purity of the obtained exosomes. The distribution in the expression of miRs and its differential analysis showed a significant increase in miR-376a-3p as well as a reduction in miR-20a-5p in patients vs. healthy controls. Functional analysis of miR-376a-3p determined its interaction with gene targets involved in autophagy (ATG4C), TGF-b signaling (ACVR1C) and cell survival (PIK3R1, IGF1R). Protein levels of these substrates indicated the potential control of TGF-beta signaling pathway by the downregulation of ACVR1C, and the increase in survival pathway measured by IGFR1 presence in patient’s serum. Conclusion CD patients show a higher number of circulating exosomes, with an overexpression of miR-376a-3p. The involvement of this microRNA in different intracellular signaling pathways suggests its participation in the unbalance of the immune activity present in CD.