Identification and verification of disulfidptosis-related genes in sepsis-induced acute lung injury

Abstract

Background: Sepsis-induced acute lung injury (ALI) is a common and serious complication of sepsis that eventually progresses to life-threatening hypoxemia. Disulfidptosis is a newly discovered type of cell death associated with the pathogenesis of different diseases. This study investigated the potential association between sepsis-induced acute lung injury and disulfidptosis by bioinformatics analysis.

Methods: In order to identify differentially expressed genes (DEGs) linked to sepsis, we screened appropriate data sets from the GEO database and carried out differential analysis. The key genes shared by DEGs and 39 disulfidptosis-related genes were identified: ACSL4 and MYL6 mRNA levels of key genes were detected in different datasets. We then used a series of bioinformatics analysis techniques, such as immune cell infiltration analysis, protein-protein interaction (PPI) network, genetic regulatory network, and receiver operating characteristic (ROC), to investigate the possible relationship between key genes and sepsis. Then, experimental verification was obtained for changes in key genes in sepsis-induced acute lung injury. Finally, to investigate the relationship between genetic variants of MYL6 or ACSL4 and sepsis, Mendelian randomization (MR) analysis was applied.

Results: Two key genes were found in this investigation: myosin light chain 6 (MYL6) and Acyl-CoA synthetase long-chain family member 4 (ACSL4). We verified increased mRNA levels of key genes in training datasets. Immune cell infiltration analysis showed that key genes were associated with multiple immune cell levels. Building the PPI network between MYL6 and ACSL4 allowed us to determine that their related genes had distinct biological functions. The co-expression genes of key genes were involved in different genetic regulatory networks. In addition, both the training and validation datasets confirmed the diagnostic capabilities of key genes by using ROC curves. Additionally, both in vivo and in vitro experiments confirmed that the mRNA levels of ACSL4 and MYL6 in sepsis-induced acute lung injury were consistent with the results of bioinformatics analysis. Finally, MR analysis revealed a causal relationship between MYL6 and sepsis.

Conclusion: We have discovered and confirmed that the key genes ACSL4 and MYL6, which are linked to disulfidptosis in sepsis-induced acute lung injury, may be useful in the diagnosis and management of septic acute lung injury.

Keywords: Mendelian randomization; disulfidptosis; immune infiltration; network analysis; sepsis.

Figure 1

Flowchart of this study. The primary flowchart used in this experimental design of this study. DRGs, disulfidptosis-related genes; DEGs, differentially expressed genes; ROC, receiver operating characteristic; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; miRNA, microRNA.

Figure 2

Identification of DEGs and disulfidptosis-related DEGs. Volcano plot of DEGs in the GSE26378 (A), GSE28750 (B), and GSE65682 (C) datasets between control groups and sepsis patients. DEGs with downregulated expression are indicated by blue dots, whereas DEGs with upregulated expression are indicated by red dots. Venn diagram of the downregulated genes (D) and upregulated genes (E) in GSE26378, GSE28750, and GSE65682; (F) Venn diagram between DEGs and DRGs.

Figure 3

Key genes mRNA levels in training datasets. The mRNA levels of ACSL4 (A–C) and MYL6 (D–F) in the sepsis group and the control group were detected in the GSE26378, GSE28750, and GSE65682 datasets, respectively.

Figure 4

Immune cell infiltration and correlation analysis. (A) Relative abundance of 22 types of infiltrating immune cells between the sepsis and the control groups; (B) Correlation heatmap of 22 types of immune cells; (C) Boxplot of immune cells between the sepsis and the control groups; (D) Heatmap of correlations between immune cells and key genes. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

PPI network and function prediction for the key genes and their related genes (A) PPI network of ACSL4 and MYL6; (B) GO enrichment and KEGG pathways analyses of related genes using the Sankey bubble chart. BP, Biological process; CC, cellular component; MF, molecular function; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 6

Identification and network analysis of overlapping genes between ACSL4 co-expression genes and DEGs. (A) Venn diagram showing the overlapping genes between ACSL4 co-expression genes and DEGs in sepsis; (B) PPI network of the discovered overlapping genes; (C) TF-miRNA coregulatory interactions of discovered overlapping genes.

Figure 7

Identification and network analysis of overlapping genes between MYL6 co-expression genes and DEGs. (A) Venn diagram showing the overlapping genes between MYL6 co-expression genes and DEGs in sepsis; (B) PPI network of the discovered overlapping genes; (C) TF–miRNA coregulatory interactions of discovered overlapping genes.

Figure 8

Performance of the key genes in the diagnosis of sepsis in the training datasets. ROC curves of the two key genes in GSE26378 (A), GSE28750 (B), and GSE65682 (C) datasets. The AUC of MYL6 and ACSL4 in different datasets is shown.

Figure 9

Key genes mRNA levels and ROC analysis in the validation dataset. (A,B) ACSL4 and MYL6 mRNA expressions in sepsis and control groups were detected in the GSE95233 dataset, respectively; (C) ROC curves of the two key genes in the GSE95233 dataset.

Figure 10

mRNA levels of key genes in the mouse model of sepsis-induced ALI and in the LPS-treated RAW264.7. (A) HE staining in the Sham group and the LPS group; (B) Lung injury score in the Sham group and the LPS group; mRNA levels of TNF-α (C), IL-6 (D), ACSL4 (E), and MYL6 (F) in the mouse model; The mRNA levels of TNF-α (G), IL-6 (H), ACSL4 (I), and MYL6 (J) in the cell model.

Figure 11

Level of disulfidptosis in the mouse model of sepsis-induced ALI and in the LPS-treated RAW264.7. The G6P content (A), G6PDH activity (B), and NADP+/NADPH ratio (C) in the mouse model; The G6P content (D), G6PDH activity (E), NADP+/NADPH ratio (F) in the cell model.