Effect of ferroptosis-related genes on immune infiltration and prognosis in hepatocellular carcinoma
10.3760/cma.j.cn121382-20250616-00046
- VernacularTitle:铁死亡相关基因对肝细胞癌免疫浸润及预后的影响
- Author:
Wenjie ZHANG
1
;
Yuxin LI
;
Hongyan JIANG
;
Lina MAO
;
Yue MA
;
Qiangsong WANG
;
Yuanyuan ZHAO
Author Information
1. 中国医学科学院 北京协和医学院生物医学工程研究所，天津 300192
- Keywords:
Ferroptosis;
Hepatocellular carcinoma;
Prognostic risk signature;
Differentially expressed genes
- From:
International Journal of Biomedical Engineering
2025;48(4):377-391
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish ferroptosis-related risk characteristics, to evaluate the prognostic correlation of ferroptosis-related genes in hepatocellular carcinoma, and to explore the complex relationship between hepatocellular carcinoma, ferroptosis and immune microenvironment.Methods:The bioinformatics analysis involved obtaining ferroptosis-related differentially expressed genes (DEGs) from the GeneCards database and the cancer genome atlas database. The biological functions of ferroptosis-related DEGs were analyzed using gene ontology and Kyoto encyclopedia of genes and genomes pathway enrichment. Ferroptosis-related DEGs clusters were identified using univariate Cox regression analysis and cluster analysis, etc. The correlation between ferroptosis-related DEGs clusters and tumor immune microenvironment and tumor occurrence score was evaluated using immunopanoramic analysis and tumor-related score analysis. Based on ferroptosis-related characteristics, a ferroptosis-related characteristic spectrum and nomogram were constructed using multivariate Cox regression and correlation analysis, etc. The correlation between the risk characteristics and tumor immune microenvironment, tumor occurrence score and gene mutation were evaluated using immune panoramic analysis, tumor-related score analysis and gene mutation analysis. In the experimental verification stage, the mRNA expression levels of aurora kinase A ( Aurka), acetyl-CoA carboxylase alpha ( Acaca) and arrestin domain containing 3 ( Arrdc3) in mouse primary hepatocytes and mouse hepatoma Hepa1-6 cells were verified by real-time reverse transcription-PCR (RT-qPCR). The mRNA expression levels of AURKA, ACACA and ARRDC3 in adjacent normal tissues and tumor tissues of patients with hepatocellular carcinoma were verified by RT-qPCR. A heat map was used to show the correlation between clustering and clinical parameters, and this was analyzed using a chi-square test. Significance analysis was performed using a two-sided unpaired t test. Results:A total of 35 up-regulated genes and 19 down-regulated genes were identified. These genes were mainly involved in biological processes and signaling pathways related to ferroptosis, oxidative stress and fatty acid metabolism. A total of 14 ferroptosis-related DEGs were identified to be associated with prognosis. The clusterring effect was best when hepatocellular carcinoma patients were divided into two subgroups. The survival rate of cluster 2 was lower than that of cluster 1 ( P<0.05). There was no significant difference in the tumor immune dysfunction and exclusion (TIDE) score between cluster 2 and cluster 1 ( P=0.43). Cluster 1 exhibited higher levels of immune cell infiltration, particularly CD4 + T cells ( P<0.01). The expression levels of 10 major histocompatibility complex (MHC) molecule-related genes were higher in cluster 1. The angiogenesis activity score ( P=0.048) and stemness score ( P=0.038) of cluster 2 were increased, and the expression levels of programmed death-1 ( PDCD1) and cytotoxic T lymphocyte-associated antigen-4 ( CTLA-4) in cluster 2 (5.924±0.013 and 5.475±0.042) were higher than those in cluster 1 (4.539±0.143 and 4.372±0.176) (both P<0.05). The expression levels of AURKA, glucose-6-phosphate dehydrogenease ( G6PD), ACACA, GABA type A receptor associated protein like 1 ( GABARAPL1) and ARRDC3 were correlated with the T stage, clinical stage and survival status of hepatocellular carcinoma. The survival rate of the high-risk group was lower than that of the low-risk group with time ( P<0.01). The area under the curve of the risk characteristics at 1, 3 and 5 years was 0.797, 0.717 and 0.639, respectively. The actual survival time 1, 3, and 5 years was highly consistent with the corresponding predicted survival time. The levels of memory B cell infiltration, angiogenesis activity score and cell stemness score, programmed death-ligand 1, CTLA-4, hepatitis A virus cell receptor 2, lymphocyte activation gene 3 and PDCD1 gene expression (0.013 8±0.036 0, 0.884±0.212, 0.387±0.135, 6.273±0.228, 5.847±0.331, 8.179±0.259, 6.859±0.263 and 5.142±0.326) in the high-risk group were higher than those in the low-risk group (0.001 5±0.021 0, 0.874±0.132, 0.298±0.125, 5.866±0.132, 3.742±0.237, 7.236±0.321, 6.324±0.242 and 4.513±0.211) ( P<0.05, 0.01). The expression levels of MHC molecule-related genes in the high-risk group were also higher than those in the low-risk group ( P<0.05, 0.01), while the infiltration levels of resting mast cells, activated natural killer cells, and resting natural killer cells (0.043 2±0.135 0, 0.032 1±0.143 0 and 0.016 3±0.001 9) and the TIDE score (0.072 0±0.018 0) in the high-risk group were lower than those in the low-risk group (0.054 9±0.023 0, 0.042 7±0.017 0, 0.024 6±0.021 2 and 0.094 0±0.013 5) ( P<0.05, 0.01). The top five genes with the highest mutation frequency in the high-risk group were tumor protein P53 ( TP53, 43%), titin ( TTN, 21%), catenin beta 1 ( CTNNB1, 20%), mucin 16 ( MUC16, 18%) and piccolo presynaptic cytomatrix protein ( PCLO, 11%). The top five genes with the highest mutation frequency in the low-risk group were CTNNB1 (30%), TTN (24%), albumin ( ALB, 16%), MUC16 (15%) and PCLO (11%). The cube protein and PCLO showed the co-occurrence of gene mutations in the high-risk group, while MUC16 and axis 1 protein showed the co-occurrence of gene mutations in the low-risk group. There was no significant difference in tumor mutation burden (TMB) between the high-risk group (1.374±0.026) and the low-risk group (1.303±0.081) ( P=0.073). There was no significant difference in survival time between the high-TMB group (2.3 years) and the low-TMB group (3.8 years) ( P=0.293). The mutation rates of AURKA, G6PD, ACACA, GABARAPL1 and ARRDC3 genes (2.0%, 2.0%, 4.0%, 0.3% and 0.6%) were relatively low. The relative expression levels of Aurka, Acaca and Arrdc3 mRNA in Hepa1-6 cells (13.331±0.000, 6.619±0.000 and 1.209±0.002) were higher than those in mouse primary hepatocytes (1.000±0.000, 1.000±0.000 and 1.000±0.000) (all P<0.01). The relative expression levels of AURKA, ACACA and ARRDC3 mRNA in tumor tissues of patients with hepatocellular carcinoma (2.102±0.365, 2.476±0.351 and 11.460±9.189) were higher than those in adjacent normal tissues of patients with hepatocellular carcinoma (1.122±0.648, 0.831±0.935 and 0.852±0.171) ( P<0.05, 0.01). Conclusions:This study constructed a prognostic signature comprising five ferroptosis-related genes ( AURKA, G6PD, ACACA, GABARAPL1, and ARRDC3) that is highly correlated with clinical hepatocellular carcinoma data. This study highlights the significance of ferroptosis-related genes as prognostic markers for hepatocellular carcinoma and provides insights into the complex relationship between hepatocellular carcinoma, ferroptosis, and the immune microenvironment.
