As obligate intracellular parasites, viruses must rely on host cell for replication, and its infection also cause changes in the morphology and function of host cells. SARS-CoV–2 is a monopartite, single-stranded, and positive-sense RNA virus with a genome size of 29,903 nucleotides, making it the second-largest known RNA genome 15. Severe COVID–19 was characterized by respiratory failure caused by hyper-inflammation, which not only bring great difficulties to clinical treatment, but also increase mortality for disease 16. It is now well established that a cytokine storm syndrome (CSS) involved in molecules like interleukin 6 (IL6), interleukin 8 (IL8), E-cadherin, MCP–1, and VEGF, probably may be the reason for hyper-inflammation in severe cases 2,17.
Investigating the differences in genetic level during the disease occurrence and development not only help us understand disease more clearly, but also guide for developing countermeasures. In the present study, candidate datasets were consisted of two parts, in vivo level (patient-data) and in vitro level (NHBE-data and A549 data), and they were annotated by the same platform of GPL18573 [Illumina NextSeq 500 (Homo sapiens)], which eliminated the adverse effect of the bias of gene coverage and algorithm usage from different sequencing platforms on the results and conclusion. After completing the differential analysis and GSEA, we found that in samples with SARS-CoV–2 infection, inflammation-related hallmark gene sets like IL6-Jak-Stat3-signaling, IL2-Stat5-signaling, complement, coagulation, angiogenesis, Kras-signaling-up, and TGF-- signaling were activated while cell cycle-related hallmark gene set like E2F-targets was suppressed both in vivo and vitro level. Subsequently, inflammation-related genes were found in the core genes of those co-enriched gene sets, such as IL6, ICAM1, IL1B, CSF1, TLR2, VEGFA, TNF, and so on. We constructed PPI network for the co-core genes, and analyzed molecular modules through MCODE, and identified two key molecular modules. Module one consisted of 17 genes: BCL2A1, BCL2L1, SOD2, PTGS2, TNF, PLAUR, MYC, LIF, VEGFA, LOX, IL6, CSF1, C3, TLR2, ICAM1, IL1B, and TNFAIP3, which were mainly involved in inflammatory response pathway and complement system cascade, such as such as TNF signaling, IL–17 signaling, PI3K-Akt signaling, Jak-Stat signaling, NOD-like receptor signaling, Toll-like-receptor signaling, and complement signaling. Module two consisted of 9 genes: EZH2, PTTG1, DLGAP5, RFC3, DUT, RPA2, CCNB2, PCNA, and MCM4, were mainly involved in cell cycle and DNA replication signaling. Finally, by assessing the level of gene expression in the patient-data, we found genes from module one showed a relatively high-level expression while genes from modules two showed low-level. All of these findings may provide novel evidences selecting effective mRNA biomarkers to evaluate disease progression and predict prognosis for patients with SARS-CoV–2 infection. Also, these findings improve our understanding of additional targets for anti-SARS-CoV–2 agents.
Cascade reactions of inflammation and immunity are the two key aspects in the pathogenic process of virus infecting the host, which often requires multiple cells and cellular components to participate, and lead to changes in the level of gene expression. It is no exception for SARS-CoV-2. GSEA, based upon functional class scoring methods, is capable of solving biological problem by focusing on gene sets rather than individual genes 11. In our study, we did use this method for analyzing genetic changes related to inflammation and immune response after SARS-CoV–2 infection, which preserved gene-gene correlations, and provided an improved understanding of the biological functional enrichment in the groups of high-level and low-level expressed genes 18. For hallmarks gene sets, we found two gene sets involved in two significant molecules in the interleukin super family, gene set of IL6-Jak-Stat3-signaling (genes were up-regulated by IL6 via Stat3 during acute phase response) and IL2-Stat5-signaling (genes up-regulated by Stat5 in response to IL2 stimulation), were activated in samples of SARS-CoV–2 infection. Several studies have been reported that IL6, as one of the genes involved in cytokine release syndrome (CRS), was used to assess severity of COVID 19 like respiratory failure, ARDS, and adverse clinical outcomes 19–24. Similar to these conclusions, we identified IL6 not only was at a high-level expression in data of cell lines and cases died due to SARS-CoV–2 infection, but also interacted the most with other genes in the PPI network, which confirmed again its value of as an active cytokine with a wide range of biological functions and as an effective biomarker for the prognosis of COVID 19. IL2 is a secreted cytokine that was important for the proliferation of T and B lymphocytes25,26. Here, no core role of IL2 was observed even though we found activated IL2- Stat5-signaling in the infected samples, but IL2-related genes with the example of CSF1 and LIF, were among the co-core genes with a high-level expression, which revealed that the action of IL2 might be as a ‘homeostatic’ cytokine, and mainly involved in the immune response but not in CRS. Moreover, the gene sets of TNFA-signaling (genes regulated by NF-κB in response to TNF) and Kras-signaling-up (genes up-regulated by Kras activation) were also activated in patients with COVID 19, which further uncover the roles of genes and cytokines such as NF-κB, TNF, and Kras in the pathogenesis and progression of SARS-CoV–2 infection. These factors together with IL6 might lead to the possibility of severe and/or fatal status for COVID 19. Notably, gene set of epithelial-mesenchymal-transition (EMT) (genes defining EMT) was represented in activated state in SARS-CoV–2 infected samples, which implied the potential of SARS-CoV–2 to lead to pulmonary interstitial fibrosis (PIF) as the disease progress. PIF was also an important feature of COVID 19 cases with poor prognosis, as previously reported 27,28.Apart from this, gene set of complement (genes encoding components of the complement system, which is part of the innate immune system) and gene set of coagulation (genes up-regulated during formation of blood vessels) were also found as activated status in our result, which indicated that the disease evolution of SARS-CoV–2 infection was a multiple pathway, complicated process, comprising of various dynamic changes in the genome.
E2F transcription factors play critical roles in the control of transcription, cell cycle and apoptosis29–31. For samples with SARS-CoV–2 infection, we found that the gene set of E2F targets was suppressed obviously, which indicated that cell cycle disorders might occur in COVID 19.
From a clinical perspective, this might be related to hypoxia caused by SARS-CoV–2 infection in human bodies 32, because hypoxia could directly affect cell differentiation and energy metabolism 33. From gene level, we identified that genes like PCNA, CCNB2, PTTG1, as were at a low-level expression in the samples of SARS-CoV–2 infection. According to literature report, PCNA and PTGG1 involved in regulating the biological function of p21 that was a CDK inhibitor to trigger cell-cycle arrest in the G1 and G2 phases 34,35. CCNB2 involved in the formation of CCNB2/CDK1 complex that controlled separase activity through inhibition of phosphorylation and regulated the biological process of G2 phase of cell cycle 36. Incorporating previous studies, we proposed that in the process of SARS-CoV–2 infecting the host, genetic changes caused cell cycle disorders, which might manifest as hyper-active S-phase and hypo-active G2 phase facilitating viral DNA replication.
Other co-core genes, such as ICAM1, a leukocyte adhesion molecule, mainly encoded a cell surface glycoprotein typically expressed on endothelial cells and immune cells. As reported, ICAM1 plays an important role in enhancing CD16+ monocyte adhesion to the endothelium 37, and regulating IL6/Akt/Stat3/NF-κB-dependent pathway 38. For roles in viral infection, ICAM1 had been determined to involve in DC-mediated HIV–1 transmission to CD4(+) T cells 39 and regulate interferon-gamma and IL17 in hepatitis B virus infection 40. Our results showed that ICAM1 participated in the signaling pathways of NF-κB, IL17, and TNF, which was in line with similar previous studies. TNFAIP3, induced by the TNF, plays a role in inhibiting the activation of NF-κB as well as TNF-mediated apoptosis. Literature data reported TNFAIP3 was closely associated with the replication of viruses like influenza A 41, and hepatitis B virus 42. In our study, we identified TNFAIP3, TNF, and IL1B were involved in the signaling pathway of necroptosis.
Similar to apoptosis, necroptosis was executed via distinctive signaling mechanism comprising a cascade of specified proteins, resulting in regulated necrotic cell death 43. Physiologically, necroptosis induced an innate immune response as well as premature assembly of viral particles in cells infected with virus that abrogates host apoptotic machinery, which was advantageous for the host. On the other hand, necroptosis was also deleterious because it can cause various diseases such as sepsis, neurodegenerative diseases and ischemic reperfusion injury 43. Qin et al. observed that necroptosis of the pulmonary epithelium was associated with severe H7N9 infection leading to ARDS, and the final conclusion indicated that necroptosis inhibition might be a novel therapy for H7N9 influenza virus 44. However, the benefits of necroptosis to the host may sometimes be outweighed by the potentially deleterious hyperinflammatory consequences of activating this death modality in pulmonary and other tissues45,46. All this evidence remined us that necroptosis signaling was a double-edged sword in the defense to microbial infection, or even might be the likely culprit of the critical and/or lethal SARS-CoV–2 infected cases. This also provided a novel clue for necroptosis signaling in the treatment of COVID 19 and served it as a potential therapeutic target. BCL2A1, one of the pro-inflammatory cytokines, encoded a member of the BCL–2 protein family. In our study, we found BCL2A1 participated in the signaling of NF-κB and apoptosis, which were similar to previous reports in the literature 47,48. Nevertheless, more details of how BCL2A1 affected the pathogenesis of SARS-CoV–2 infection still needed to be explored.
For signaling pathway, besides mentioned above, signaling pathway of AGE-RAGE, MAPK, cytokine-cytokine receptor interaction, EGFR tyrosine kinase inhibitor resistance, viral protein interaction, were enriched in our results. From these findings, we believed that in the course of SARS-CoV–2 infection, the dysregulation was gene-specific rather than pathway-specific. Hence, we tried to construct molecular modules not only for accurate prediction but also for the evaluation of the effects of genes on COVID 19 patients’ prognosis. Our data indicated that in severe and/or fatal SARS-CoV–2 infection cases, immune responses tended to be gentle while inflammatory cascades were hyper-activated, which might be attributable to aberrant gene expression. However, in terms of how genes found in our results participated in the pathogenesis of SARS-CoV–2, it is still an unresolved problem, and further needed to verify in vivo data.