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. 2021 Nov 22;16(11):e0260450.
doi: 10.1371/journal.pone.0260450. eCollection 2021.

Core genes involved in the regulation of acute lung injury and their association with COVID-19 and tumor progression: A bioinformatics and experimental study

Affiliations

Core genes involved in the regulation of acute lung injury and their association with COVID-19 and tumor progression: A bioinformatics and experimental study

Aleksandra V Sen'kova et al. PLoS One. .

Abstract

Acute lung injury (ALI) is a specific form of lung damage caused by different infectious and non-infectious agents, including SARS-CoV-2, leading to severe respiratory and systemic inflammation. To gain deeper insight into the molecular mechanisms behind ALI and to identify core elements of the regulatory network associated with this pathology, key genes involved in the regulation of the acute lung inflammatory response (Il6, Ccl2, Cat, Serpine1, Eln, Timp1, Ptx3, Socs3) were revealed using comprehensive bioinformatics analysis of whole-genome microarray datasets, functional annotation of differentially expressed genes (DEGs), reconstruction of protein-protein interaction networks and text mining. The bioinformatics data were validated using a murine model of LPS-induced ALI; changes in the gene expression patterns were assessed during ALI progression and prevention by anti-inflammatory therapy with dexamethasone and the semisynthetic triterpenoid soloxolone methyl (SM), two agents with different mechanisms of action. Analysis showed that 7 of 8 revealed ALI-related genes were susceptible to LPS challenge (up-regulation: Il6, Ccl2, Cat, Serpine1, Eln, Timp1, Socs3; down-regulation: Cat) and their expression was reversed by the pre-treatment of mice with both anti-inflammatory agents. Furthermore, ALI-associated nodal genes were analysed with respect to SARS-CoV-2 infection and lung cancers. The overlap with DEGs identified in postmortem lung tissues from COVID-19 patients revealed genes (Saa1, Rsad2, Ifi44, Rtp4, Mmp8) that (a) showed a high degree centrality in the COVID-19-related regulatory network, (b) were up-regulated in murine lungs after LPS administration, and (c) were susceptible to anti-inflammatory therapy. Analysis of ALI-associated key genes using The Cancer Genome Atlas showed their correlation with poor survival in patients with lung neoplasias (Ptx3, Timp1, Serpine1, Plaur). Taken together, a number of key genes playing a core function in the regulation of lung inflammation were found, which can serve both as promising therapeutic targets and molecular markers to control lung ailments, including COVID-19-associated ALI.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig 1
Fig 1. Key genes involved in the development of Acute Lung Injury (ALI): Results of bioinformatics analysis.
(A) Venn diagram illustrating overlap between differentially expressed genes (DEGs) identified by the analysis of GSE80011, GSE130936, GSE58654, and GSE94522 datasets of ALI caused by different agents. (B) Functional analysis of overlapping DEGs identified in different GSEs. Enrichment for Gene Ontology (biological processes), KEGG, REACTOME, and Wikipathways terms were performed using the ClueGo plugin in Cytoscape. The labels of the most significant terms are shown. Colours of nodes represent the term enrichment significance. Only pathways with p < 0.05 after the Bonferroni step-down correction for multiple testing were included in the networks. (C) Heat map showing expression levels of DEGs in different GSEs. Heat map construction and hierarchical clustering (Euclidean distances) were performed using Morpheus. LogFC = Log2 (fold change). (D) Heat map demonstrating the interconnection of DEGs in a protein-protein interaction network (PPI) reconstructed for each ALI dataset using the STRING database (confidence score ≥ 0.7, maximal number of interactors = 0) in Cytoscape. Degree: number of interactions between a DEG and its partners. Inflammatome: number of interactions between DEGs and partners in the rodent inflammatome, constructed based on transcriptomic data published by Wang et al. [54]. (E) Co-occurrence of identified DEGs with relevant keywords in the scientific literature deposited in the MEDLINE database. Analysis was performed using the GenClip3 web service. Data were visualised via Circos.
Fig 2
Fig 2. Soloxolone Methyl (SM) and dexamethasone (Dex) effectively ameliorate Acute Lung Injury (ALI) in vivo.
(A) The chemical structures of SM and Dex. (B) The experimental setup. SM (10 mg/kg) and Dex (1 mg/kg) were administered to mice 1 h prior to LPS instillation (10 mg per mice), followed by the evaluation of ALI 24 h after the induction of lung inflammation. (C) Total (left) and differential (right) leukocyte counts in the bronchoalveolar (BAL) fluid of healthy and LPS-challenged mice receiving SM or Dex therapy. Total leukocyte counts were estimated using a Neubauer chamber. The distribution of leukocyte subpopulations was measured by microscopy after staining of cells with azur-eosin by Romanovsky-Giemsa. (D, E) The levels of pro-inflammatory cytokines (TNF-α and IL-1β) in the lung tissue measured by qRT-PCR and BAL fluid measured by ELISA. (F) Representative histological images of lung sections of healthy and LPS-challenged mice receiving SM or Dex therapy. Haematoxylin and eosin staining, original magnification ×100 (upper panel), periodic acid-Schiff staining, original magnification ×400 (middle panel), and immunohistochemical staining with anti-TNF-α primary antibodies, original magnification ×400 (bottom panel). The black arrows indicate inflammatory infiltration, the green arrows indicate mucus hyperproduction, and red arrows indicate TNF-α hyperexpression in lung tissue.
Fig 3
Fig 3. Involvement of ALI-associated DEGs in the regulation of lung injury induced by SARS-CoV-2 infection in humans.
(A) Upper panel: Venn diagram of genes differentially expressed in the lungs injured by various irritants (ALI) or SARS-CoV-2 (COVID-19) in mice and humans, respectively. Lower panel: The heat map illustrating the expression of 15 overlapping genes between ALI and COVID-19 in analysed datasets. (B) The degree centrality values of ALI-associated DEGs in COVID-19-related gene association network. (C) The network of 15 overlapping genes between ALI and COVID-19 with their first neighbours within the COVID-19-related regulome. (D) Functional annotation of the network depicted in Fig 3C, using the ClueGO plugin in Cytoscape. (E) Expression of the top 5 ALI-associated DEGs, i.e. those most interconnected with the COVID-19-related regulome, in the lungs of healthy mice or mice with LPS-induced ALI without treatment and after SM or Dex administration. Relative expression levels of these genes were normalised to the expression level of hypoxanthine phosphoribosyltransferase (HPRT) (used as a reference gene). Three samples from each experimental group were analysed in triplicate. The data are shown as mean ± standard deviation. * p<0.05, ** p<0.01, *** p<0.001, n.s.—not significant. The red bar represents changes in the expression of the mentioned genes in the lungs of COVID-19 patients vs. healthy group, according to Daamen et al. [82]. (F) The expression levels and the degree centrality values of mice-specific ALI-related core genes in human ALI/ARDS induced by different stimuli.
Fig 4
Fig 4. Association of identified DEGs with survival of patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC).
(A) Survival analysis of DEGs was performed using The Cancer Genome Atlas (TCGA) data for patients with LUAD and LUSC. Visualisation was performed via Circos. The width of ribbons corresponds with the log-rank p-value, with wider ribbons indicating a more significant correlation. The ribbons of ELN and CAT indicate that low expression of these genes is associated with poor survival of patients, as opposed to the other genes, for which high expression is associated with poor survival. The absence of ribbons indicates no significant correlation between gene expression and patient survival. (B) Survival of patients with LUAD and LUSC depending on the level of particular gene expression in the lung tissue (mRNA level). Kaplan-Meier survival curves were constructed based on TCGA data using OncoLnc. (C) Survival of patients with lung cancers depending on the level of particular gene expression in the lung tissue (protein level). Kaplan-Meier survival curves were constructed based on The Human Protein Atlas data.
Fig 5
Fig 5. Interconnections and interplay of revealed key ALI-associated genes in different pulmonary disorders.
(A) The heatmap showing interconnections of revealed ALI-associated key genes with the rodent inflammatome, the COVID-19 regulome, and the progression of lung cancer in patients. Degree: the number of linkages of the gene with partner genes within the gene association network. In the case of LUAD/LUSC, only those genes are marked in which the expression level is significantly and negatively associated with overall survival time for patients with lung cancer. (B) The hierarchical network reconstructed with all revealed key regulatory genes involved in ALI development (STRING, confidence score ≥ 0.7).

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References

    1. Feng X, Jia A. Protective Effect of Carvacrol on Acute Lung Injury Induced by Lipopolysaccharide in Mice. Inflammation. 2014;37: 1091–1101. doi: 10.1007/s10753-014-9833-1 - DOI - PubMed
    1. Lee JW, Chun W, Kwon OK, Park HA, Lim Y, Lee JH, et al.. 3,4,5-Trihydroxycinnamic acid attenuates lipopolysaccharide (LPS)-induced acute lung injury via downregulating inflammatory molecules and upregulating HO-1/AMPK activation. Int Immunopharmacol. 2018;64: 123–130. doi: 10.1016/j.intimp.2018.08.015 - DOI - PubMed
    1. Zhang H, Chen S, Zeng M, Lin D, Wang Y, Wen X, et al.. Apelin-13 Administration Protects Against LPS-Induced Acute Lung Injury by Inhibiting NF-κB Pathway and NLRP3 Inflammasome Activation. Cell Physiol Biochem. 2018;49: 1918–1932. doi: 10.1159/000493653 - DOI - PubMed
    1. Yang J, Li S, Wang L, Du F, Zhou X, Song Q, et al.. Ginsenoside Rg3 attenuates lipopolysaccharide-induced acute lung injury via MerTK-dependent activation of the PI3K/AKT/mTOR pathway. Front Pharmacol. 2018;9. doi: 10.3389/fphar.2018.00850 - DOI - PMC - PubMed
    1. Bellani G, Laffey JG, Pham T, Fan E, Brochard L, Esteban A, et al.. Epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries. JAMA—J Am Med Assoc. 2016;315: 788–800. doi: 10.1001/jama.2016.0291 - DOI - PubMed

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This research was supported by the Russian Science Foundation (grant #19-74-30011) and the Russian State funded budget projects of ICBFM #0245-2021-0004. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.