Whole-transcriptome analysis of Aortic Stenosis reveals dysregulated RNA networks, immune cell infiltration, and NADK2 as a candidate regulator

Aortic stenosis (AS) is a common cardiovascular condition marked by progressive narrowing of the aortic valve and is associated with substantial morbidity and mortality. Although its clinical impact is well recognized, the molecular mechanisms driving AS progression remain incompletely defined, particularly with regard to the contributions of non-coding RNAs and immune–metabolic interactions. High-throughput RNA sequencing combined with integrative bioinformatics analyses was performed on human aortic valve tissues from patients with AS to identify differentially expressed RNAs. Expression of Nicotinamide Adenine Dinucleotide Kinase 2 (NADK2) was validated using western blotting and quantitative polymerase chain reaction in both human AS specimens and murine AS models. Functional enrichment analyses, immune cell infiltration profiling, and weighted gene co-expression network analysis were applied to characterize regulatory networks and identify hub genes. A total of 1,443 messenger RNAs (mRNAs), 3,147 long non-coding RNAs (lncRNAs), and 145 circular RNAs (circRNAs) were differentially expressed in AS valve tissues, with the majority showing increased expression. Principal component analysis demonstrated clear separation between AS and control samples. Functional enrichment analyses linked differentially expressed mRNAs primarily to sensory perception and calcium signaling pathways, lncRNAs to embryonic development–related processes, and circRNAs to protein regulatory functions. NADK2 was identified as a hub gene and was significantly upregulated at both the mRNA and protein levels in valve tissues from patients with AS and in the murine AS model. Immune infiltration analysis indicated increased proportions of CD4⁺ memory T cells and CD8⁺ T cells in AS, with strong positive correlations observed between these immune cell populations and NADK2 expression. Weighted gene co-expression network analysis further supported NADK2 as a key candidate regulator within the disease-associated magenta module. This study delineates comprehensive RNA regulatory networks in patients with AS and identifies NADK2 as a key molecular contributor to disease pathogenesis. Its consistent upregulation across species, together with close associations with immune dysregulation and co-expression network modules, supports its potential relevance as a therapeutic target in AS.