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Hepatitis B virus (HBV) X protein (HBx) is critical in hepatocellular carcinoma (HCC) development, but its influence on tumor immunity and the tumor microenvironment (TME) remains unclear. This study aimed to construct a prognostic model based on HBx-related genes and explore their relationship with immune infiltration and immunotherapy response. Through transcriptome sequencing of our HBx-expressing HepG2 cells and analysis of HCC patient data from the cancer genome atlas (TCGA) and genotype-tissue expression (GTEx), we identified seven HBx-related genes, nuclear VCP-like (NVL), WD repeat domain 75 (WDR75), NOP58 nucleolar protein (NOP58), Brix domain-containing protein 1 (BRIX1), deoxynucleotidyltransferase terminal interacting protein 2 (DNTTIP2), MKI67 FHA domain interacting nucleolar phosphoprotein (NIFK), and ribosome production factor 2 (RPF2), associated with poor prognosis. LASSO Cox regression narrowed these to four key genes (BRIX1, RPF2, DNTTIP2, and WDR75), which were used to develop a prognostic riskscore signature. High-risk patients exhibited lower survival rates, decreased infiltration of anti-tumor immune cells, poorer responses to immunotherapy, and increased immune evasion. Among the four genes, DNTTIP2 showed higher expression in single-cell data, was linked to migration inhibitory factor (MIF) signaling, and may play a pivotal role in shaping an immunosuppressive TME. Elevated DNTTIP2 expression was confirmed in HBx-expressing HepG2 cells and HBV-infected HCC samples. This study highlights a novel HBx-related four-gene prognostic model that predicts clinical outcomes, immune infiltration, and immunotherapy response, offering insights into HCC progression and potential therapeutic targets.

乙型肝炎病毒（Hepatitis B virus, HBV）X蛋白（HBx）在肝细胞癌（hepatocellular carcinoma, HCC）发生发展中发挥关键作用，但其对肿瘤免疫及肿瘤微环境（tumor microenvironment, TME）的影响仍不明确。本研究旨在构建基于HBx相关基因的预后模型，并探讨其与免疫浸润及免疫治疗应答的关联。本研究通过对转染HBx的HepG2细胞进行转录组测序，并分析来自癌症基因组图谱（The Cancer Genome Atlas, TCGA）及基因型-组织表达（Genotype-Tissue Expression, GTEx）的肝细胞癌患者数据，最终筛选出7个与不良预后相关的HBx相关基因：核VCP样蛋白（nuclear VCP-like, NVL）、WD重复结构域75（WD repeat domain 75, WDR75）、核仁蛋白NOP58（NOP58 nucleolar protein, NOP58）、含Brix结构域蛋白1（Brix domain-containing protein 1, BRIX1）、脱氧核苷酸转移酶末端相互作用蛋白2（deoxynucleotidyltransferase terminal interacting protein 2, DNTTIP2）、MKI67 FHA结构域相互作用核仁磷酸蛋白（MKI67 FHA domain interacting nucleolar phosphoprotein, NIFK）及核糖体生成因子2（ribosome production factor 2, RPF2）。随后通过LASSO Cox回归分析将上述基因缩减为4个核心基因（BRIX1、RPF2、DNTTIP2及WDR75），并以此构建预后风险评分模型。高风险组患者生存率更低、抗肿瘤免疫细胞浸润水平下降、免疫治疗应答更差且免疫逃逸程度更高。在这4个核心基因中，DNTTIP2在单细胞测序数据中表达量更高，且与迁移抑制因子（migration inhibitory factor, MIF）信号通路相关，可能在塑造免疫抑制性肿瘤微环境中发挥关键作用。研究团队还在转染HBx的HepG2细胞及HBV感染的肝细胞癌样本中验证了DNTTIP2的高表达。本研究提出了一种全新的HBx相关四基因预后模型，该模型可有效预测临床结局、免疫浸润水平及免疫治疗应答，为肝细胞癌的发病机制研究及潜在治疗靶点开发提供了新的科学见解。