Single-cell transcriptomics identifies PRDX1 and GPX4 as potential ferroptosis regulators and tumor...

The present study aimed to investigate the expression and roles of peroxiredoxin 1 (PRDX1) and glutathione (GSH) peroxidase 4 (GPX4) in ferroptosis regulation and the tumor microenvironment in hepatocellular carcinoma (HCC). The expression levels of PRDX1 and GPX4 were assessed in HCC through analysis of datasets from The Cancer Genome Atlas (TCGA), GSE14520 from the Gene Expression Omnibus database and the Human Protein Atlas, along with HCC cell lines. Diagnostic and prognostic values were evaluated using receiver operating characteristic curves and Kaplan-Meier survival analyses. The single-cell dataset GSE149614 was employed to investigate the cellular localization and characteristics of specific cell subpopulations expressing PRDX1 or GPX4 within the HCC tumor microenvironment. The relationships between PRDX1, GPX4 and ferroptosis-related pathways were explored using the FerrDb database and Gene Set Enrichment Analysis. Additionally, their interactions with the immune microenvironment were examined utilizing the CIBERSORT algorithm. To validate the results of the bioinformatics analysis, the present study utilized small interfering (si)RNA technology to construct single gene knockdown and dual gene co-knockdown models targeting PRDX1 and GPX4 in Hep3B cells. Subsequently, the proliferative activity of each group of cells was evaluated using the Cell Counting Kit-8 method and the levels of the intracellular lipid peroxidation product malondialdehyde (MDA) were detected using the MDA detection kit. The results of the present study demonstrated that the expression levels of PRDX1 and GPX4 were upregulated in HCC tissues and cell lines. The area under the curve for the combined diagnostic model of PRDX1 and GPX4 in HCC was 0.83. Furthermore, elevated expression levels of PRDX1 or GPX4 predicted reduced overall survival (OS), and their expression was weakly positively correlated in HCC. Single-cell transcriptomics demonstrated predominant expression of both genes in malignant cells. GPX4 was primarily detected in early-differentiation subgroups enriched in metabolic and ferroptosis pathways, whereas PRDX1 was predominantly expressed in intermediate-differentiation subgroups associated with antioxidative stress pathways. The dual-high PRDX1/GPX4 expression group exhibited increased reactive oxygen species, GSH and iron levels, whereas the dual-low group had prolonged OS. Immune infiltration analysis indicated a trend of higher M0 macrophage levels and lower CD4⁺ T-cell counts in the dual-high expression group. Furthermore, PRDX1 expression was positively correlated with multiple immune checkpoint genes, whereas GPX4 expression was markedly negatively correlated only with CD274 and TIGIT. The siRNA-PRDX-1/siRNA-GPX4-1 group exhibited significantly inhibited HCC cell proliferation and promoted MDA production; however, proliferative activity was partially increased compared with that in the siRNA-PRDX-1 group, whereas the levels of MDA accumulation were significantly reduced compared with in the siRNA-GPX4-1 group. In conclusion, the current study revealed the concerted co-expression of PRDX1 and GPX4 in malignant HCC, which was associated with ferroptosis evasion and immune suppression. Furthermore, the present findings suggested that PRDX1 may shift from an antioxidant to a pro-oxidant mediator under GPX4 deficiency, thereby alleviating lipid peroxidation. These findings support targeting this axis as a future therapeutic breakthrough.