The impact of inflammation and iron metabolism on gene expression alterations in ischemic stroke: a bioinformatics approach

Figures

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  Fig. 1. Flow Chart for the Comprehensive Analysis of IIMRDEGs. IS, Ischemic Stroke; GSEA, Gene Set Enrichment Analysis; DEGs, Differentially Expressed Genes; IRGs, Inflammatory-Related Genes; IMRGs, Iron-Metabolism-Related Genes; IIMRGs, Inflammatory&Iron-Metabolism-Related Genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; IIMRDEGs, Inflammatory&Iron-Metabolism-Related Differentially Expressed Genes; PPI, Protein-Protein Interaction Network; ROC, Receiver Operating Characteristic; TF, Transcription Factor.

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  Fig. 2. Batch Effects Removal of GSE22255 and GSE16561. (A) Box plot of Combined GEO Datasets distribution before batch removal. (B) Post-batch integrated GEO datasets (combined datasets) distribution box plots. (C) PCA plot of the datasets before debatching. (D) Go to the PCA map of the combined GEO datasets after batch processing. PCA, Principal Component Analysis; IS, Ischemic Stroke. GSE16561 is the IS dataset in light green, and GSE22255 is the IS dataset in light brown.

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  Fig. 3. Differential Gene Expression Analysis. (A) Volcano plot of differentially expressed gene analysis of IS samples and control samples in the combined GEO datasets. (B) Venn diagram of DEGs and IIMRGs in the integrated GEO datasets (combined datasets). (C) Heatmap of IIMRDEGs in the integrated GEO datasets (combined datasets). IS, ischemic stroke; DEGs, differentially expressed genes; IIMRGs, inflammation and iron metabolism-related genes; IIMRDEGs, inflammation and iron metabolism-related differentially expressed genes. IS samples are shown in orange, and control samples are shown in light gray. In the heatmap, red represents high expression and blue represents low expression.

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  Fig. 4. GO and KEGG Enrichment Analysis for IIMRDEGs. (A) Bar graph of GO and KEGG enrichment analysis results of IIMRDEGs : BP, CC, MF and KEGG. B. Bubble diagram of GO and KEGG enrichment analysis results of IIMRDEGs : BP, CC, MF and KEGG. GO terms and KEGG terms are shown on the ordinate. C-F. GO and KEGG enrichment analysis results of IIMRDEGs: BP (C), CC (D), MF (E) and KEGG (F). Yellow nodes represent items, blue nodes represent molecules, and the lines represent the relationship between items and molecules. The bubble size in the bubble plot represents the number of genes, while the color of the bubbles indicates the size of the p value. Redder colors correspond to smaller p values, whereas bluer colors correspond to larger p values. The screening criteria for GO and KEGG enrichment analysis were p value < 0.05 and FDR (q value) < 0.25, with the p value correction method being Benjamini-Hochberg (BH).

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  Fig. 5. GSEA for Combined Datasets. (A) GSEA mountain map presentation of biological functions in the combined GEO datasets. B-E. GSEA showed that all genes were significantly enriched in NF-κB activation through the FADD-RIP1 pathway mediated by caspase 8 and 10 (B), TRAF6-mediated NF-κB activation (C), the NF-κB pathway (D), and the NF-κB canonical pathway (E). The screening criteria for GSEA were p < 0.05 and FDR (q-value) < 0.25, with the p-value correction method being Benjamini-Hochberg (BH).

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  Fig. 6. PPI Network and hub genes Analysis. PPI network of IIMRDEGs calculated from the STRING database (A). (B–F) The PPI network of the top 10 IIMRDEGs, calculated using five algorithms from the CytoHubba plug-in, including Maximal Clique Centrality (MCC) (B), Density of Maximum Neighborhood Component (DMNC) (C), Degree (D), Closeness (E), and Betweenness (F). G. Venn diagram of the top 10 IIMRDEGs from the five algorithms of the CytoHubba plug-in. H. GeneMANIA website predicts the interaction network of the eight hub genes with similar functions. The circles in the figure represent the hub genes and genes with similar functions, while the colors corresponding to the lines indicate the interconnected functions.

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  Fig. 7. Regulatory Network of IIMRDEGs. (A) mRNA-TF Regulatory Network of hub genes. (B) mRNA-miRNA Regulatory Network of hub genes. Orange is mRNA, green is TF, and blue is miRNA.

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  Fig. 8. Differential Expression Validation and ROC Curve Analysis. (A) Group comparison plots of hub genes in IS samples and control samples from the combined GEO datasets. ROC curves for hub genes IL7R and LCN2 (B), HLA-DQB1 and ADM (C), PTGS2 and IL1B (D), and DUSP1 and MMP9 (E) in the integrated GEO datasets (combined datasets) are displayed. The symbols denote statistical significance: * indicates p < 0.05 (statistically significant); ** represents p < 0.01 (highly statistically significant); and *** represents p < 0.001 (very highly statistically significant). When AUC > 0.5, it indicates that the expression of the molecule is trending towards promoting the occurrence of the event, and the closer the AUC is to 1, the better the diagnostic effect. AUC demonstrates low accuracy in the range of 0.5 to 0.7, and moderate accuracy in the range of 0.7 to 0.9. TPR, true positive rate; FPR, false positive rate. Light gray represents control samples, while orange represents IS samples.

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  Fig. 9. Combined Datasets Immune Infiltration Analysis by CIBERSORT Algorithm. (A,B) The proportion of immune cells in the integrated GEO datasets is shown in a bar graph (A) and a group comparison graph (B). (C) Correlation heatmap of immune cell infiltration abundance in the integrated GEO datasets. (D) Bubble plot illustrating the correlation between hub genes and immune cell infiltration abundance in the integrated GEO datasets. The symbols indicate statistical significance: * represents p < 0.05 (statistically significant); ** represents p < 0.01 (highly statistically significant); and *** represents p < 0.001 (very highly statistically significant). An absolute value of the correlation coefficient (r) below 0.3 indicates weak or no correlation, while a correlation between 0.5 and 0.8 is considered moderate. IS samples are depicted in orange, and control samples are shown in light gray. Blue indicates negative correlation, while red indicates positive correlation, with color depth representing the strength of the correlation.