Single‐Cell Sequencing and Mendelian Randomization Reveal T Cell Nuclear Factor Genes in Hepatocellular Carcinoma Progression

This study integrates single‐cell RNA sequencing with Mendelian randomization to elucidate the role of nuclear factor of activated T cells (NFAT)‐related genes in the progression of hepatocellular carcinoma (HCC). The GSE162616 dataset was analyzed to identify differentially expressed cells and NFAT‐related genes through quality control, clustering, and z ‐score algorithms. Mendelian randomization analysis of expression quantitative trait loci (eQTL) data was performed to identify hub genes causally linked to HCC. Validation in The Cancer Genome Atlas‐Liver Hepatocellular Carcinoma cohort included survival analysis, clinical correlation, and nomogram construction. Sixteen cell clusters were resolved and annotated into five types: natural killer (NK) cells, T cells, B cells, hepatocytes, and monocytes. Differentially expressed NFAT‐related genes were predominantly enriched in immune and cytokine pathways. Three genes—CACYBP, CTLA4, and RGCC—were identified as causally associated with HCC and designated as hub genes. T cells and NK cells emerged as key cellular populations, and pseudotime analysis delineated T cell differentiation trajectories. Cell–cell communication analysis revealed robust interactions between NK and B cells and between NK and T cells, primarily via the MIF–(CD74+CXCR4) axis. All three hub genes were upregulated in HCC tissues. A nomogram integrating these genes exhibited excellent diagnostic performance (AUC = 0.9). These results establish CACYBP and RGCC as risk factors and CTLA4 as a protective factor for HCC. The nomogram offers a potential tool for early diagnosis and immunotherapy guidance. Our findings highlight the value of integrating single‐cell transcriptomics with Mendelian randomization for prioritizing causal genes and provide novel insights into NFAT‐mediated immune regulation in HCC.