Abstract
BACKGROUND: Pulmonary hypertension (PH) is a severe progressive disease characterised by elevated pulmonary vascular resistance and right ventricular hypertrophy. Increasing evidence has highlighted the vital role of nicotinamide adenine dinucleotide (NAD(+)) metabolism in cardiovascular disease. However, the role of NAD(+) metabolism-related genes (NMRGs) in PH remains unclear. In this study, we aimed to identify novel NMRGs as biomarkers in PH. METHODS: Using the Gene Expression Omnibus database and Limma R package, we identified differentially expressed genes (DEGs) of PH and downloaded NMRGs from Kyoto Encyclopedia of Genes and Genomes and Reactome databases. Candidate NMRGs were subsequently identified by overlapping DEGs, NMRGs, and module genes obtained by weighted gene co-expression network analysis. The diagnostic value of these candidate NMRGs was evaluated using receiver operator characteristic (ROC) curve analysis, and gene set enrichment analysis (GSEA) was performed to explore the functional roles of hub genes. CIBERSORT algorithm was employed to assess immune cell infiltration in the PH microenvironment. Finally, the functional role of target genes in PH was validated through in vitro cellular experiments. RESULTS: Through comprehensive bioinformatics analyses across multiple datasets, we identified two NMRGs: NT5E and CD38. ROC analysis confirmed the higher predictive accuracy of NT5E, with area under the ROC curve values reaching 0.891 and 0.894 in GSE113439 and GSE53408 datasets, respectively. GSEA revealed that patients with high NT5E expression exhibited significant enrichment of PH-related biological functions and pathways. Given the relationship between NAD(+) and immunity, immune infiltration analysis was performed, which showed a close association between NT5E expression and plasma cells and eosinophils in the PH microenvironment. In vitro experiments further showed that NT5E was significantly upregulated in a PH cell model, and knockdown of NT5E attenuated hypoxia-induced proliferation, resistance to apoptosis, and migratory ability of pulmonary arterial smooth muscle cells. CONCLUSION: Based on bioinformatics analysis and in vitro validation, we confirmed that NMRGs affect PH progression, with NT5E showing potential as a novel diagnostic marker and therapeutic target in PH.