Bioinformatics analysis to investigate the potential relationship between mitochondrial structure and function-related genes and the immune microenvironment in atherosclerosis

Abstract

Objective: This study aims to elucidate the interactions between genes associated with mitochondrial structure and function and the immune microenvironment in atherosclerosis.

Methods: Differentially expressed mitochondria-related genes (DE-MRGs) were identified through the analysis of two gene expression datasets, GSE100927 and GSE159677, in conjunction with a list of mitochondria-related genes sourced from the MitoCarta3.0 database. The immune profile of infiltrating immune cells in atherosclerotic carotid artery (CA) patients compared to controls (CTLs) was assessed using CIBERSORT. Potential target genes were screened based on Spearman correlation analysis between specific DE-MRGs and differentially expressed immune cells. Furthermore, the correlation between characterized DE-MRGs and immune cells in AS was examined at the single-cell level, and the expression of key genes was validated in vitro.

Results: Our study identified a robust association between four key genes-C15orf48, UCP2, PPIF, and MGST1-among 15 DE-MRGs, and immune macrophage polarization. These genes exhibited alterations corresponding to the degree of macrophage differentiation in AS. Additionally, Gene Set Enrichment Analysis (GSEA) revealed that C15orf48, UCP2, PPIF, and MGST1 modulate multiple immune pathways within the body. The mRNA expression levels of these four key genes in AS were confirmed via quantitative real-time PCR (qRT-PCR), with results aligning with bioinformatics predictions. Compared to the control group, the expression levels of C15orf48, UCP2, and PPIF were significantly elevated in AS macrophages, whereas MGST1 expression was notably reduced in AS macrophages. Consequently, these mitochondria-related genes-C15orf48, UCP2, PPIF, and MGST1-may influence the immune microenvironment in AS by modulating macrophage differentiation.

Conclusion: C15orf48, UCP2, PPIF, and MGST1 may serve as potential therapeutic targets for enhancing the atherosclerotic immune microenvironment in future interventions.

Keywords: atherosclerosis; immune microenvironment; inflammation; macrophage; mitochondria; single-cell analysis.

Figure 1

Workflow diagram. The flowchart of this study.

Figure 2

Screening of differentially expressed mitochondria-associated genes. (A) Differential mitochondria-associated pathways between CA/CTL samples; (B) volcano plot of differentially expressed genes between CA/CTL samples; (C) heatmap of differentially expressed genes between CA/CTL samples; (D) Wayne's plot of the intersection of MRGs and DEGs; and (E) heatmap of the correlation of DE-MRGs with mitochondrial pathways.

Figure 3

DE-MRG and immune cell correlation analysis. (A) Box line plot of immune cell proportions; (B) box line plot of immune cell proportion differences; (C) heatmap of correlations between key genes and differential immune cells. *represents P < 0.05, **represents P < 0.01, ***represents P < 0.001, ****represents P < 0.0001; red represents a positive correlation, and blue represents a negative correlation.

Figure 4

Single-cell data analysis. (A) Characteristic violin plots of single-cell sequencing data; (B) inflection point plots; (C) cell clustering-UMAP; (D) cell marker expression heatmap; (E) manually annotated cell subpopulations; and (F) histograms of the proportions of single-cell subpopulations.

Figure 5

Expression analysis of DE-MRGs in single cells. (A) Single-cell differential gene volcano map; (B) single-cell subpopulation immune-related pathway scoring heatmap; (C) single-cell subpopulation mitochondrial pathway scoring heatmap; and (D) single-cell subpopulation key gene expression point map.

Figure 6

Macrofitting time series analysis. (A–C) Macromimetic time series analysis; (D) histogram of cell proportions in different branches; (E) scatter plot of key gene expression in different states.

Figure 7

Key gene function prediction. (A) C15orf48-KEGG enrichment ridge map; (B) MGST1-KEGG enrichment ridge map; (C) PPIF-KEGG enrichment ridge map; (D) UCP2-KEGG enrichment ridge map.

Figure 8

Expression detection of key genes in AS macrophages (A) statistical graph of C15orf48 mRNA expression level; (B) statistical graph of PPIF mRNA expression level; (C) statistical graph of MGST1 mRNA expression level; (D) statistical graph of UCP2 mRNA expression level.