IER3 as an RFX5 transcriptional co-activator promotes hepatocellular carcinoma progression via AKR1B10-mediated p53 transcriptional regulation

INTRODUCTION: Hepatocellular carcinoma (HCC) exhibits profound molecular heterogeneity, which complicates prognosis and therapy. Identifying key molecular subtypes and their driving oncogenes is crucial for developing targeted strategies. This study aimed to delineate chemokine-based HCC subtypes and investigate the functional role and mechanism of a critical identified driver, Immediate Early Response 3 (IER3). METHODS: Bioinformatic analysis of public datasets was performed for molecular subtyping, differential expression, survival, and pathway enrichment. Clinical relevance was validated via immunohistochemistry on a tissue microarray containing 48 paired HCC/adjacent tissues. In vitro functional assays (CCK-8, colony formation, flow cytometry, wound healing, and Transwell) were conducted in SMMC-7721 cells following siRNA-mediated knockdown. Mechanistic studies employed dual-luciferase reporter, co-immunoprecipitation, qPCR, and western blot to delineate the IER3/RFX5/AKR1B10/p53 axis. RESULTS: Unsupervised clustering based on chemokine expression stratified HCC into two subtypes: an immune-active subtype (Cluster 1) enriched in interferon-γ signaling and a matrix-remodeling subtype (Cluster 2). Prognostic analysis further refined three patient clusters, with the aggressive Subtype C exhibiting worst survival (HR = 3.78, p < 0.001) and significant IER3 upregulation. Clinical samples confirmed IER3 protein overexpression correlated with advanced grade (p < 0.001). In vitro, IER3 knockdown suppressed proliferation, colony formation, migration/invasion, and induced apoptosis. Mechanistically, IER3 functioned as a transcriptional coactivator for RFX5, forming a complex that upregulated AKR1B10, which subsequently mediated the suppression of the tumor suppressor p53. CONCLUSION: This study defines a chemokine-based duality in HCC and identifies IER3 as a key driver of the aggressive subtype. Our findings indicate that IER3 contributes to HCC malignancy through the novel IER3/RFX5-AKR1B10-p53 axis in vitro, positioning it as a potential therapeutic target warranting further in vivo investigation.