Deregulated miRNA expression is associated with endothelial dysfunction in post-mortem lung biopsies of COVID-19 patients

Abstract

MicroRNAs (miRNAs) are critical modulators of endothelial homeostasis, which highlights their involvement in vascular diseases, including those caused by virus infections. Our main objective was to identify miRNAs involved in the endothelial function and determine their expression in post-mortem lung biopsies of COVID-19 patients with severe respiratory injuries and thrombotic events. Based on functional enrichment analysis, miR-26a-5p, miR-29b-3p, and miR-34a-5p were identified as regulators of mRNA targets involved in endothelial and inflammatory signaling pathways, as well as viral diseases. A miRNA/mRNA network, constructed based on protein-protein interactions of the miRNA targets and the inflammatory biomarkers characterized in the patients, revealed a close interconnection of these miRNAs in association to the endothelial activation/dysfunction. Reduced expression levels of selected miRNAs were observed in the lung biopsies of COVID-19 patients (n = 9) compared to the Controls (n = 10) (P < 0.01-0.0001). MiR-26a-5p and miR-29b-3p presented the best power to discriminate these groups (area under the curve (AUC) = 0.8286, and AUC = 0.8125, respectively). The correlation analysis of the miRNAs with inflammatory biomarkers in the COVID-19 patients was significant for miR-26a-5p [IL-6 (r² = 0.5414), and ICAM-1 (r² = 0.5624)], and miR-29b-3p [IL-4 (r² = 0.8332) and IL-8 (r² = 0.2654)]. Altogether, these findings demonstrate the relevance and the non-random involvement of miR-26a-5p, miR-29b-3p, and miR-34a-5p in endothelial dysfunction and inflammatory response in patients with SARS-CoV-2 infection and the occurrence of severe lung injury and immunothrombosis.

Keywords: COVID-19; SARS-CoV-2; endothelial dysfunction; lung injuries; microRNA.

Figure 1.

A: heatmap of KEGG pathways for the selected three miRNAs (DIANA tools v.5.0). Black boxes indicate the pathways with relevance to the endothelial function and to viral infection diseases. The color intensities indicate the −log10 (P value) enrichment score of each Gene Ontology (GO) term. B: miRNA/mRNA targets network. In red, green, and blue are the direct targets of miR-26a-5p, 29b-3p, and miR-34a-5p, respectively. In grey, the inflammatory biomarkers analyzed in the group of patients and in orange the SARS–CoV-2 S protein receptors ACE2 and TMPRSS2. Black arrows link each miRNA to its putative targets, and grey lines link the interaction of all the genes (Cytoscape 3.0).

Figure 2.

A–C: relative expression of miRNAs between the Control (C) and COVID-19 (CV) groups. D–J: ROC/AUC analysis of individual and combined miRNAs between the Control and COVID-19 groups. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 3.

A: the endothelial dysfunction process in the alveolar capillary membrane of SARS–CoV-2 infected alveoli. B: correlation analysis (r²) of the miR-26a-5p and IL-6 expression levels in the COVID-19 group. C: higher expression of IL-6 levels in the COVID-19 group compared to the Controls (Mann-Whitney test, ***P = 0.0001). D: IL-6 tissue expression (IHC) positivity in the endothelium of the lung vessel (star) and in type II pneumocytes (arrowhead) of a representative tissue section of a COVID-19 sample, compared to the negative expression of lung vessels in a Control sample (black arrow). C and D were constructed based on the data described in Nagashima et al. (3).