Altered microRNA expression in severe COVID‐19: Potential prognostic and pathophysiological role

BackgroundThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is ongoing. The pathophysiology of SARS-CoV-2 infection is beginning to be elucidated but the role of microRNAs (miRNAs), small non-coding RNAs that regulate gene expression, remains incompletely understood. They play a role in the pathophysiology of viral infections with potential use as biomarkers. The objective of this study was to identify miRNAs as biomarkers of severe COVID-19 and to analyze their role in the pathophysiology of SARS-CoV-2 infection. MethodsmiRNA expression was measured in nasopharyngeal swabs from 20 patients with severe COVID-19, 21 patients with non-severe COVID-19 and 20 controls. Promising miRNAs to differentiate non-severe from severe COVID-19 patients were identified by differential expression analysis and sparse Partial Least Squares-Discriminant Analysis (sPLS-DA). ROC analysis, target prediction, GO enrichment and pathway analysis were used to analyze the role and the pertinence of these miRNAs in severe COVID-19. ResultsThe number of expressed miRNAs was lower in severe COVID-19 patients compared to non-severe COVID-19 patients and controls. Among the differentially expressed miRNAs between severe COVID-19 and controls, 5 miRNAs were also differentially expressed between severe and non-severe COVID-19. sPLS-DA analysis highlighted 8 miRNAs, that allowed to discriminate the severe and non-severe COVID-19 cases. Target and functional analysis revealed enrichment for genes involved in viral infections and the cellular response to infection as well as one miRNA, hsa-miR-15b-5p, that targeted the SARS-CoV-2 RNA. The comparison of results of differential expression analysis and discriminant analysis revealed three miRNAs, namely hsa-miR-125a-5p, hsa-miR-491-5p and hsa-miR-200b-3p. These discriminated severe from non-severe cases with areas under the curve ranging from 0.76 to 0.80. ConclusionsOur analysis of miRNA expression in nasopharyngeal swabs revealed several miRNAs of interest to discriminate severe and non-severe COVID-19. These miRNAs represent promising biomarkers and possibly targets for antiviral or anti-inflammatory treatment strategies.

and Drosha was significantly down-regulated in COVID-19 patients. 4 Furthermore, miRNA depletion enhances proinflammatory cytokine production, including expression of interleukin-6 (IL-6). 5 The global repression of miRNA expression in severe COVID-19 that we observed here may thus be causally linked to the hyperinflammatory state found in severe COVID-19.
The comparison of results of differential expression analysis and sPLS-DA revealed three miRNA in common, namely hsa-miR-125a-5p, hsa-miR-491-5p and hsa-miR-200b-3p ( Figure 1B, panel b). These miRNAs discriminated severe from non-severe cases with areas under the curve ranging from 0.76 to 0.79 ( Figure 2). To evaluate their performance as biomarkers to predict disease severity, it would be interesting to determine their expression at different time points, that is, before, during and after resolution of severe COVID-19.
We hypothesized that the 10 miRNAs selected by sPLS-DA and/or differential analysis ( Figure 1B) play a role in the pathophysiology of severe COVID-19. Ninety-five validated target genes were retrieved (Table S3). Gene ontology (GO) enrichment analysis revealed enrichment for five, eight and zero GO terms associated with biological process, molecular function and cellular component, respectively   (Tables S4 and S5). Enriched biological processes were involved in deoxyribonucleid acid (DNA) damage, ubiquitination and antigen processing and presentation (Table  S4). Enriched molecular functions were involved in protein kinase activity, ubiquitination and RNA polymerase II activity (Table S5). Kegg pathway analysis revealed enrichment in pathways that play a role in ubiquitination, viral infections and the immune response (Table 2). Reactome pathway analysis showed enrichment for pathways involved in antigen processing, NFkappaB and other signaling pathways ( Table 2).
Of the miRNAs found of interest to discriminate between severe and non-severe COVID-19 in our study, some were reported to be implicated in viral and other TA B L E 1 miRNAs differentially expressed between severe COVID-19 and controls and non-severe COVID-19 infections: miR-455-5p was up-regulated in rabies virus infection in vitro, decreased suppressor of cytokine signaling 3 (SOCS3) expression and increased signal transducer and activator of transcription 3 (STAT3) activity, resulting in enhanced viral replication and the production of IL-6. 6 Furthermore, this miRNA targeted the C-C motif chemokine receptor 5 (CCR5). 7 Of interest, CCR5 is involved in severe COVID-19 and has been proposed as anti-inflammatory treatment target. 8 Taken together, this underlines the role of hsa-miR-455-5p in viral infections and the inflammatory response and its potential as target of therapeutic interventions. hsa-miR-532-3p diminished the levels of ASK1 and downstream phosphorylation/translocation of p38 MAPK during lipopolysaccharide (LPS)/ tumor necrosis factorα (TNF-α)-induced inflammation in macrophages and reduced the release of various pro-inflammatory cytokines and chemokines, including IL-6 and TNF-alpha. 9 IL-6 is a proinflammatory cytokine that has been reported to be involved in the cytokine storm observed in severe COVID-19. 10 The down-regulation of anti-inflammatory miRNAs, such as hsa-miR-532-3p, hsa-miR-340-5p and hsa-miR-455-5p, in severe COVID-19 (Table 1) is in line with a hyperinflammatory state in severe COVID-19. Supplementation of these anti-inflammatory miRNAs may represent a novel therapeutic strategy.
Recently, direct and indirect miRNA interactions with other miRNAs have been described (reviewed 11 ). Therefore, it cannot be excluded that the miRNAs identified in this study directly or indirectly influence expression of other miRNAs and thus have a broad impact on miRNA and gene expression.
We next searched for miRNAs that may directly target the SARS-CoV-2 genome among the miRNAs associated with severe COVID-19. We found that hsa-miR-15b-5p was predicted to bind to the SARS-CoV-2 genome at 16 positions (Table S6). Furthermore, hsa-miR-15b-5p was confirmed to interact with SARS-CoV-2 RNA in vitro. 12 hsa-miR-15b-5p was down-regulated in severe COVID-19 in our study (Figure 1B,panel c) and in the lungs of hamsters infected with SARS-CoV-2. 13 Taken together, this suggests that the down-regulation of hsa-miR-15b-5p may represent a mechanism of SARS-CoV-2 to escape the host antiviral defense.
In conclusion, our analysis of miRNA expression in nasopharyngeal swabs revealed a general reduction of miRNA expression in severe COVID-19 patients. Several miRNAs of interest to discriminate severe and nonsevere COVID-19 were identified. Functional analysis of these miRNAs suggested a role in the pathophysiology of the disease. Further characterization of their implication in SARS-CoV-2 infection will enable elucidation of the molecular mechanisms and may reveal potential targets for antiviral or anti-inflammatory treatment of COVID-19.