Integrated bioinformatics and in vitro validation reveal TUBB as a core gene and therapeutic target in NAFLD-related hepatocellular carcinoma.

Non-alcoholic fatty liver disease (NAFLD) is a major cause of chronic liver disease worldwide and is closely associated with the development of hepatocellular carcinoma (HCC), yet the molecular mechanisms underlying this transition remain poorly understood. In this study, transcriptomic datasets of NAFLD and HCC were obtained from TCGA, ICGC, and GEO, and differentially expressed genes were identified and integrated using weighted gene co-expression network analysis. Functional enrichment, protein-protein interaction analysis, and multiple machine learning algorithms were employed to determine core candidate genes. The diagnostic and prognostic significance of key genes was evaluated through ROC and survival analyses, while immune infiltration characteristics were assessed using CIBERSORT. Single-cell RNA sequencing was further used to explore cell-type-specific expression patterns. Potential therapeutic agents targeting the core gene were screened via DGIdb, validated by molecular docking, and subsequently examined through in vitro assays. A total of 37 shared genes were identified, primarily enriched in pathways related to zinc ion homeostasis, copper detoxification, and apoptosis. β-Tubulin (TUBB) was identified as a pivotal biomarker exhibiting strong diagnostic, prognostic, and immunological relevance. Moreover, three clinically used drugs targeting TUBB demonstrated inhibitory effects on HCC cell proliferation, migration, and invasion in vitro. These findings suggest that TUBB may serve as both a diagnostic biomarker and a promising therapeutic target in the progression from NAFLD to HCC, offering potential avenues for early detection and targeted treatment.