Integrative bulk and single-cell transcriptome analyses reveal mitochondria and immune-related biomarkers in atherosclerosis with experimental validation

Background The development of atherosclerosis (AS) is associated with mitochondrial function and immunity, yet the intrinsic mechanisms involved remain insufficiently elucidated. Therefore, the objective of the study was to explore mitochondria- and immune-related biomarkers in AS via single-cell RNA sequencing (scRNA-seq) and bulk RNA analysis. Methods In this study, transcriptomic and scRNA-seq data of AS, mitochondria-related genes (MRGs), and immune-related genes (IRGs) were obtained from public databases. The research team pinpointed candidate genes by conducting both differential expression analysis and weighted gene co-expression network analysis (WGCNA). To identify potential biomarkers, they employed protein-protein interaction (PPI) analysis, machine learning techniques, and verified expression levels. Following these initial discoveries, the investigators carried out enrichment and immune infiltration analyses to assess how these biomarkers might contribute to the regulatory mechanisms of AS. Moreover, scRNA-seq analysis was performed to identify key cells. Ultimately, the reverse transcription quantitative PCR (RT-qPCR) was employed to validate the expression levels of biomarkers in clinical specimens. Results The research successfully identified five biomarkers for AS, namely PLCβ4, RAC2, TYROBP, VAV1, and VCL. The dilated cardiomyopathy pathway showed positive enrichment in PLCβ4 and VCL, but negative enrichment in RAC2, TYROBP, and VAV1. Furthermore, 10 differential immune cells (DICs) were discerned between the AS and control groups. Seven DICs demonstrated notable correlations with five biomarkers, such as naive B cells. The scRNA-seq analysis identified seven distinct cell types, with macrophages, endothelial cells, and vascular smooth muscle cells (VSMCs) defined as the key cells. Finally, the RT-qPCR analysis revealed that the mRNA expression levels of RAC2, TYROBP, and VAV1 were significantly increased in the AS group, while PLCβ4 expression was markedly reduced. Conclusion This study integrated bulk and scRNA-seq analyses to identify five candidate biomarkers, of which four were experimentally validated, along with three key cell types, providing novel insights into the molecular mechanisms and potential therapeutic targets for AS.