Growing interest on miRNAs as potential biomarkers or as therapeutic drugs has raised over the last few years, especially in cancer, cardiovascular or neurodegenerative diseases. In this regard, miRNAs may have greater relevance in the future, since they provide us with a better understanding of the pathogenic mechanisms involved in different diseases. Although numerous publications concerning infection by SARS-CoV-2 have been reported in the last year, very few studies addressed its relationship with miRNAs alterations. In silico analyses have focused on prediction of miRNAs targeting SARS-CoV-2 genome to find alternative therapies [12, 23]. The present study describes a specific miRNA signature in the plasma of COVID-19 patients. A total of 15 miRNAs (7 upregulated and 8 downregulated in COVID-19), with common expression in human plasma and validated in an independent cohort appeared dysregulated in COVID-19 patients compared to CAP patients, revealing important differences in the pathophysiology of these two clinical entities despite their similarities in terms of respiratory symptoms. Due to these differences, we were able to develop a multivariate logistic regression model based on miRNAs that efficiently distinguishes patients with COVID-19 from patients with CAP.
Our study provides experimental evidence that confirms previous in silico bioinformatic analyses of possible miRNAs interacting with SARS-CoV-2 genome or playing a role in the host response to the virus. Particularly, dysregulation of miR-424-5p, miR-146a-5p, miR-130a-3p, miR-25-3p, miR-27b-3p and miR-425-5p was predicted in other studies, but it was not verified experimentally . Among the miRNAs identified in our study, miR-335-5p was significantly downregulated in COVID-19 vs. CAP patients. This miRNA has been previously associated with suppression of inflammatory processes . Its repression during SARS-CoV-2 infection may contribute to the widely described general proinflammatory status . In line with these observations, we found low levels of plasma miR-146a-5p in COVID-19 vs. CAP patients. miR-146a regulates inflammation by targeting TNF receptor associated factor 6 (TRAF6), therefore reducing expression of NF-kB [27, 28]. Moreover, decreased levels of miR-146a have been linked to higher risk of thrombotic events and neutrophil NETosis .
GO enrichment analysis revealed a potential involvement of vascular system biology in this pathology, specifically angiogenesis and response to endothelial damage. This is consistent with several articles that report atherosclerotic plaques, prothrombotic changes in endothelium, increased intussusceptive angiogenesis and a subsequent enhanced risk of thrombosis [30, 31]. EGFR is a protein involved in a great number of biological processes; some of them related to blood vessel growth, inflammation via NF-kB or profibrotic and atherosclerotic events. In this context, EGFR, a known target of miR-27b-3p, miR-146a-5p, miR-16-5p, miR-335-5p and miR-30a-5p, is found at higher levels in patients with COVID-19, likely enhancing these events [32–34].
Intriguingly, we observed increased levels of the chemokine CXCL12 in CAP vs. COVID-19 patients. CXCL12, which is the CXCR4 ligand, is necessary for effective hematopoiesis, T cell and memory B cell homing to the lymph nodes or monocyte recruitment. Inhibition of this axis is used by several viruses in order to increase their proliferation by reducing the number of circulating immune cells . Whether low levels of this chemokine in COVID-19 patients could be triggered by SARS-CoV-2 is a possibility that merits further exploration. miR-146a-5p, miR-221-3p and their target CXCL12 (according to miRTarBase), were downregulated in COVID-19 patients. This observation suggests either non-canonical regulation by these miRNAs or the prevalence of other regulatory mechanisms modulating the expression and secretion of CXCL12. Moreover, CXCL12 was found at higher levels in the plasma of severe vs. mild COVID-19 patients. In our cohort, 3 severe COVID-19 patients, with higher levels of CXCL12, had sepsis or bacterial superinfection. Thus, we cannot rule out the relationship between higher levels of this chemokine and the presence of superinfection or sepsis in critically ill COVID-19 patients, since CXCL12 has a role in neutrophil recruitment from bone marrow .
IL-17 was found at higher levels in patients with severe COVID-19 disease, in agreement with a prior study indicating its crucial role in the pathogenesis of acute respiratory distress syndrome (ARDS) . IL-17 induces recruitment of neutrophils to the lung, exacerbating proinflammatory cytokine release and leading to ARDS. Therefore, the IL-17 signalling pathway could be a potential target to treat the cytokine storm observed in the most severe COVID-19 cases . Another well-studied cytokine in the pathogenesis of this disease is IL-10. Several published works show an increase of IL-10 in critically ill COVID-19 patients despite its general anti-inflammatory nature, even proposing this protein as a prognostic biomarker [39, 40], together with other well-known inflammatory cytokines such as IL-6. Here, we report an increased amount of IL-10 in COVID-19 vs. CAP patients. Conversely, no differences were observed between mild and severe disease. Finally, IL-21 is involved in antiviral defence by enhancing Th1 and IFN-γ responses . Likewise, its role for a correct B cell memory differentiation and germinal centre reaction leading to effective antibody response could be decisive for viral clearance . Thus, low levels of its receptor, IL-21R, in severe COVID-19 could lead to an impaired IFN expression and facilitate the spread and replication of SARS-CoV-2. Receptor proteins like IL-21R or EGFR may be found in plasma by leakage from different cells and tissues. However, caution should be taken when interpreting these results, since soluble EGFR may act as a regulator of its signalling pathway, as previously described .
On the other hand, our data revealed clear differences in TIMP-2 levels between severe and mild cases of COVID-19. One of the functions of this inhibitor of matrix metalloproteinases (MMP) is to participate in the regulation of the renewal of the extracellular matrix. Besides this role, it was proposed as an inhibitor of angiogenesis by MMP-independent mechanisms . Therefore, this protein may be participating in the response to direct endothelial damage caused by SARS-CoV-2.
Despite size sample limitation, this study conclusively proves the existence of a differential profile in circulating miRNAs between COVID-19 and CAP patients. Analysis of the identified miRNAs showed regulatory functions associated with angiogenesis and inflammation, indicating that endothelial damage and vascular compromise is definitively one of the main conditions driven by SARS-CoV-2. Here, we describe new miRNAs and soluble cytokines that may contribute to gain insight into COVID-19 pathogenic mechanisms and set the basis for the urgently needed design of novel therapeutic strategies.