肿瘤微环境相关基因CCR7在肝癌铜死亡中的作用及其作为肝癌预后标志物的探索性研究

Exploratory study on the role of tumor microenvironment-related gene CCR7 in Cuproptosis and its potential as a prognostic marker for liver cancer

目的通过转录组测序及功能基因组学分析探讨肝癌肿瘤微环境(TME)相关基因与铜死亡及肝癌预后的关系。方法生物信息学与基础实验相结合的综合研究。利用癌症基因组图谱(TCGA)数据库下载424例肝癌(LIHC)患者的表达谱和临床病理信息,评估免疫评分(ImmuneScore)和基质评分(StromalScore),分为高分组和低分组,比较后取交集筛选出差异表达基因(DEG);利用单因素Cox回归分析和蛋白质相互作用(PPI)网络分析确定核心DEG;通过R语言和Kaplan-Meier分析核心基因表达水平与肝癌患者生存时间之间的关联,利用Kaplan-Meier Plotter在线数据库验证;构建肝癌铜死亡细胞模型进行RNA-seq测序,探索铜诱导的细胞死亡过程中基因表达变化;利用体外细胞实验进行验证。结果共鉴定出1701、2041个与ImmuneScore和StromalScore相关的DEG,其中包含1134个共同上调基因和60个共同下调基因。PPI网络中主导节点前30个核心基因与单因素Cox基因进行交叉分析,得到关键免疫基因CC-趋化因子受体7(CCR7)。肝癌组织中CCR7 mRNA表达水平较正常组织高(P<0.05),且CCR7高表达肝癌患者的总生存期(OS)比低表达患者更长(P=0.003)。Elesclomol-CuCl2处理能抑制肝癌细胞存活率(P<0.001),铜死亡模型RNA-seq测序发现CCR7在诱导铜死亡发生时表达下调[|log2(FC)|=2.27,P<0.001],CCR7表达下调增强了肝癌细胞对铜死亡激动剂的敏感性(P<0.001)。结论TME相关基因CCR7可能与铜死亡的发生相关,其表达下调有助于肝癌中铜死亡过程;CCR7有望成为肝癌预后的潜在生物标志物。

ObjectiveTo explore the interplay between tumor microenvironment(TME)-associated genes,cuproptosis,and the prognosis of liver cancer through transcriptome sequencing and functional genomics analysis.MethodsEmploying a hybrid approach that integrates bioinformatics with fundamental experimental research,we utilized the TCGA database to acquireexpression profiles and clinical-pathological information from 424 liver hepatocellular carcinoma patients.We evaluated ImmuneScore and StromalScore to categorize patients into high and low groups,subsequently identifying differentially expressed genes(DEG)at the intersection of these groups.Core DEG were identified through univariate Cox regression analysis and protein-protein interaction(PPI)network analysis.The association between the expression levels of core genes and the survival time of liver cancer patients was analyzed using the R language and Kaplan-Meier analysis,and verified using the Kaplan-Meier Plotter online database.We established a cuproptosis cell model and performed RNA-seq to examine gene expression alterations during copper-induced cell death,followed by in vitro cell experiments for verification.ResultsA total of 1701 and 2041 DEG were llinked t ImmuneScore and StromalScore,respectively,encompassing 1134 commonly upregulated genes and 60 commonly downregulated genes.The top 30 core genes from the PPI network's dominant nodes were cross-referenced with univariate Cox regression results,leading to the identification of the pivotal immune gene CCR7.CCR7 mRNA expression levels were higher in hepatocellular carcinoma tissues than in normal tissues(P<0.05).Patients with high expression of CCR7 in liver cancer had a longer overall survival compared to those with low expression(P=0.003).Treatment with elesclomol-CuCl2significantly curtailed the survival of hepatocellular carcinoma cel(P<0.001).RNA-seq data from the cuproptosis model indicated a downregulation of CCR7 expression during the onset of cuproptosis[|log 2(FC)|=2.27,P<0.001],and downregulation of CCR7 expression enhanced the sensitivity of hepatocellular carcinoma cells to cuproptosis inducers.ConclusionThe TME-related gene CCR7 is implicated in cuproptosis,and its downregulation might facilitate the process in liver cancer.CCR7 holds potential as a biomarker for liver cancer prognosis.