肝癌铜死亡相关基因及其与预后、免疫浸润的关系分析

Analysis of cuproptosis-related genes in liver cancer and their association with prognosis and immune infiltration

摘要:

背景与目的 肝癌是消化道恶性肿瘤之一，发病率高、病死率高。铜死亡是一种铜依赖、新型的细胞死亡方式，继发于铜过载诱发的线粒体功能受损。铜死亡在多种肿瘤中扮演重要作用，但其与肝癌的关系尚不清楚。因此，本研究探讨铜死亡相关基因在肝癌中的表达特征，以及与肝癌预后及免疫浸润的关系。方法 从TCGA和GTEx数据库下载肝癌和正常肝脏组织转录组数据进行差异表达和突变分析。采用R语言“clusterProfiler”包进行GO和KEGG富集分析。采用LASSO、单因素和多因素回归分析筛选影响肝癌患者预后的基因并构建风险因子图。使用R包“rms”构建列线图。使用UALCAN数据库分析铜死亡相关基因与肝癌临床病理特征的关系并验证。使用Spearman相关性分析铜死亡相关基因与免疫细胞浸润和免疫检查点的相关性。采用TIMER2.0数据库分析铜死亡相关基因表达与肿瘤相关成纤维细胞（CAF）浸润的相关性，采用TISDB数据库分析CDKN2A和DLAT表达与髓源抑制性细胞（MDSC）浸润丰度的相关性。结果 与正常肝脏组织比较，9个铜死亡相关基因在肝癌中表达显著升高，CDKN2A突变频率最高。铜死亡相关基因主要参与蛋白质脂酰化、三羧酸循环、柠檬酸循环等生物过程。基于LASSO、单因素与多因素回归分析筛选出影响肝癌患者总体生存率（OS）的基因CDKN2A和DLAT，并以此构建风险因子图，时间依赖性受试者工作特征曲线显示其具有较好的预测能力。通过单因素和多因素回归分析筛选出CDKN2A、DLAT、T分期和肿瘤状态是影响肝癌患者OS的独立预后因素，基于上述因素构建了列线图，校正曲线显示该列线图预测和实际观察之间有很好的一致性。UALCAN数据库分析发现CDKN2A、DLAT与肝癌临床分期、肿瘤分级有关，且GEO数据库、HPA数据库及肝癌细胞中的验证结果与之一致。相关性分析显示，CDKN2A和DLAT表达与免疫细胞浸润和免疫检查点表达相关；TIMER2.0数据库分析显示，DLAT表达与CAF浸润明显正相关；TISDB数据库分析显示，CDKN2A和DLAT表达与MDSC浸润丰度无相关性。结论 铜死亡相关基因CDKN2A、DLAT可能是肝癌新的预后生物标志物和免疫治疗的新靶点。

Abstract:

Background and Aims Liver cancer is one of the malignant tumors of the digestive system, characterized by high incidence and mortality rates. Cuproptosis, a novel copper-dependent form of cell death, occurs as a result of mitochondrial dysfunction induced by copper overload. Cuproptosis plays a significant role in various tumors, but its relationship with liver cancer remains unclear. Therefore, this study was conducted to investigate the expression characteristics of cuproptosis-related genes in liver cancer and their association with prognosis and immune infiltration.Methods Transcriptome data of liver cancer and normal liver tissues were downloaded from TCGA and GTEx databases for differential expression and mutation analysis. The R language "clusterProfiler" package was used for GO and KEGG enrichment analysis. LASSO, univariate, and multivariate regression analyses were employed to screen genes affecting the prognosis of liver cancer patients and a risk factor graph was constructed. A nomogram was constructed using the "rms" package in R. The UALCAN database was used to analyze the relationship between cuproptosis-related genes and clinicopathologic features of liver cancer and then the findings were validated. Spearman correlation analysis was used to examine the correlation between cuproptosis-related genes and immune cell infiltration and immune checkpoint expressions. TIMER2.0 database was used to analyze the correlation between the expressions of copper cuproptosis-related genes and cancer-associated fibroblast (CAF) infiltration, while TISDB database was utilized to analyze the correlation between CDKN2A and DLAT expression and myeloid-derived suppressor cell (MDSC) infiltration abundance.Results Compared to normal liver tissue, nine cuproptosis-related genes showed significantly increased expression in liver cancer, with CDKN2A having the highest mutation frequency. These genes were mainly involved in processes such as protein lipidation, tricarboxylic acid cycle, and citric acid cycle. Based on LASSO, univariate, and multivariate regression analysis, CDKN2A and DLAT were identified as genes influencing the overall survival (OS) of liver cancer patients, and a risk factor graph was constructed. Time-dependent ROC curves indicated their good predictive ability. Univariate and multivariate regression analysis revealed that CDKN2A, DLAT, T stage, and tumor status were independent prognostic factors for OS in liver cancer patients. A nomogram was constructed based on these factors, and calibration curves demonstrated good consistency between the predicted and observed values. UALCAN database analysis found associations of CDKN2A and DLAT with clinical stage and tumor grade in liver cancer, with validation results from GEO and HPA database as well as liver cancer cells aligning with these findings. Correlation analysis indicated that the expressions of CDKN2A and DLAT were correlated with immune cell infiltration and immune checkpoint expressions. TIMER2.0 database analysis revealed a significant positive correlation between DLAT expression and CAF infiltration, while TISDB database analysis showed no correlation of CDKN2A and DLAT expression with MDSC infiltration abundance.Conclusion Cuproptosis-related genes CDKN2A and DLAT may serve as novel prognostic biomarkers and potential targets for immunotherapy in liver cancer.