Identification and validation of five hypoxia- and immune-related hub targets in atrial fibrillation.

Atrial fibrillation (AF) is the most common arrhythmia, with increasing incidence and prevalence. Hypoxia and immune response are closely related to the progression of AF. This study aims to explore hub hypoxia- and immune-related genes associated with AF. Multiple AF datasets from the Gene Expression Omnibus database were downloaded. Hypoxia- and immune-related genes associated with AF were identified by integrating differential expression analysis, weighted gene co-expression network analysis (WGCNA), and protein-protein interaction (PPI) network. Functional enrichment analysis was used to evaluate the functions of key genes. Immune cell infiltration analysis was conducted using CIBERSORT and xCell. To experimentally validate the identified hub genes, RT-qPCR was performed on whole blood samples collected from healthy controls and AF patients. A total of 13 crucial hypoxia- and immune-related genes associated with AF were identified. Among them, the expressions of IL6, JUN, FOS, FOSL2, and MAFF were significantly higher in the AF group than in the sinus rhythm (SR) group, which was further validated by RT-qPCR showing increased mRNA expression in clinical whole blood samples from AF patients. Notably, transcription factors JUN, FOS, FOSL2, and MAFF exhibited clear binding peaks at promoter region of IL6. Functional enrichment analysis by GSEA revealed that these five hub genes were simultaneously involved in the cAMP signaling pathway, and four of them (JUN, FOS, FOSL2, and MAFF) were also associated with the calcium signaling pathway, indicating a potential cAMP-dependent Ca²⁺ handling mechanism in AF pathogenesis. Immune cell infiltration analysis further showed that these genes were significantly correlated with differential infiltration of various immune cells, including gamma delta T cells, monocytes, macrophages, neutrophils, and NK cells. This study successfully identified JUN, FOS, IL6, FOSL2, and MAFF as crucial hypoxia- and immune-related genes associated with AF, providing potential new targets for the treatment of AF.