Integrated multi-omics analysis identifies AKR1B15 and SLC4A10 as prognostic candidate citrullination-related genes associated with metabolic and immune programs in hepatocellular carcinoma

Protein citrullination has been increasingly implicated in tumor progression and immune regulation, but the prognostic and cellular characteristics of citrullination-related genes (CRGs) in hepatocellular carcinoma (HCC) remain incompletely understood. We integrated bulk transcriptomic and clinical data with single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) to identify prognosis-associated CRGs, construct and evaluate a prognostic model, and explore cell type-specific biological features. Candidate genes were refined using least absolute shrinkage and selection operator (LASSO) and multivariable Cox regression. Immune infiltration, regulatory network analysis, scTenifoldKnk-based in silico perturbation, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blotting were further used for biological and experimental assessment. A two-gene signature comprising AKR1B15 and SLC4A10 was independently associated with overall survival and provided additional prognostic information beyond conventional clinical variables. High-risk tumors were associated with enhanced metabolic programs and reduced stromal and overall microenvironmental scores. scRNA-seq showed that AKR1B15 was preferentially expressed in malignant cells and associated with lipid, steroid, and xenobiotic metabolism, whereas SLC4A10 was enriched in NK/T cells, particularly in a mucosal-associated invariant T (MAIT)-like population, and was associated with immune-effector programs. In silico perturbation analysis further linked the two genes to metabolic and immune-related networks, respectively. In a single HCC ST section, the corresponding malignant and NK/T-cell compartments showed a weak negative spatial association. Experimental analyses in cell lines and paired HCC tissues further supported their differential expression. AKR1B15 and SLC4A10 represent prognostic candidate CRGs associated with distinct malignant metabolic and immune-cell programs in HCC. These findings provide a multi-omics framework for prognostic stratification and support further mechanistic and spatial validation.