Integrative Multi-Omics and Functional Validation Reveal the Role of the TACE Refractoriness-Associated Gene ATP1B3 in Hepatocellular Carcinoma.

BACKGROUND: Transarterial chemoembolization (TACE) is the standard treatment for intermediate-stage hepatocellular carcinoma (HCC), but resistance to TACE is a major clinical challenge. This study aimed to identify genes associated with TACE refractoriness and their roles in HCC progression. METHODS: Gene expression profiles from 104 HCC patients treated with TACE were analyzed using unsupervised clustering to identify molecular subtypes. Key genes associated with TACE refractoriness were identified through univariate Cox regression and lasso, with ATP1B3 emerging as a candidate. Functional annotation of ATP1B3 was conducted using KEGG, GO, and GSEA analyses, while immune profiling and immunotherapy response were compared between ATP1B3-high and ATP1B3-low groups. Single-cell RNA sequencing (scRNA-seq) was employed to explore ATP1B3 expression and its cellular interactions. In vitro functional assays validated its role in migration, invasion, cell cycle, chemotherapy sensitivity, and apoptosis. RESULTS: Unsupervised clustering revealed two distinct molecular subtypes of HCC. Cluster 1 was associated with significantly prolonged overall and recurrence-free survival, whereas Cluster 2 exhibited aggressive tumor behavior and adverse clinical outcomes. ATP1B3 was identified as a pivotal gene linked to TACE refractoriness and poor prognosis. Elevated ATP1B3 expression was strongly correlated with metabolic dysregulation, heightened tumor aggressiveness, immune evasion, and diminished therapeutic responses to TACE, sorafenib, and immunotherapy. scRNA-seq analyses demonstrated widespread ATP1B3 expression across tumor and immune cell subsets, with ATP1B3-positive HCC cells displaying enhanced interactions with immune cells. Functional assays revealed that ATP1B3 overexpression promoted tumor migration, invasion, and chemoresistance, while its silencing induced cell cycle arrest, apoptosis, and increased sensitivity to cisplatin. CONCLUSION: This study identifies TACE refractoriness-related gene ATP1B3 as a key regulator of tumor progression, immune evasion, and therapeutic resistance in HCC. These findings highlight ATP1B3 as a promising biomarker for patient stratification and a potential therapeutic target to improve clinical outcomes in HCC.