Caracterização e avaliação de conteúdo de vesículas extracelulares de pacientes com COVID-19: papel potencial de microRNAs reguladores na patogênese da doença.

Abstract

Introduction: The emergence of SARS-CoV-2 caused the COVID-19 pandemic, which has become the greatest global threat to public health of this century, causing clinical conditions ranging from asymptomatic infection to severe illness with risk of death. Extracellular vesicles (EVs) function as cellular communicators transporting various molecules within the body, including regulatory miRNAs. In COVID-19, miRNAs are involved in the deregulation of important metabolic pathways, such as inflammation and hemostasis, causing generalized disorders in the body. Objective: To isolate, characterize, and evaluate the content of miRNAs in extracellular vesicles, related to inflammatory signaling pathways, virus-host interaction, and hemostasis, of patients with a confirmed diagnosis of COVID-19. Materials and Methods: Blood samples were obtained at Hospital da Baleia, Belo Horizonte, MG, and the plasma was separated and ultracentrifuged to isolate extracellular vesicles. After characterization of EVs by nanoparticle tracking analysis (NTA), electron microscopy (TEM), and western blot (WB), the miRNAs were extracted. The quantification of miRNAs occurred by RT-PCR, in the Control (GC), Moderate Covid (CM), and Severe Covid (CS) groups, comparing them with the Control group (GC). Results: The EVs of the GC, CM and CS groups showed concentrations above 6.0 x 109 particles/ml by NTA. TEM and cryomicroscopy confirmed the mean size obtained by NTA, revealing the structure of the lipid bilayer of the vesicles. WB analysis confirmed the presence of exosome marker proteins CD9 and TSG101, validating the purity of the isolation. The miRNAs miR-500-5p, -26a-5p, -16a-5p, -27a-3p, -3646, -21-5p, Let-7b-5p, -145-5p -197-3p, -155-5p and - 4282 were selected for quantification by RT-PCR, after searching the literature and bioinformatics tools. Significant differences (p<0.05) were observed for miRNAs miR-3646, -197-3p and -21-5p, regulators of the mentioned pathways, which had their expressions increased in the CM and CS groups about to the CG. Conclusion: It was found that the miRNAs miR-3646, -197-3p and -21-5p in EVs from COVID-19 patients were overexpressed. The data suggest that miR-3646 negatively regulates ACE2 and TFPI, while miR-197-3p positively regulates the GAN gene, and negatively IL-1B and finally, miR-21-5p positively regulates the GAN gene, and negatively regulates the A20 gene, important components of the inflammatory signaling and blood coagulation pathways.