Single-cell transcriptomics reveals over-activated reactive oxygen species pathway in hepatocytes in the development of hepatocellular carcinoma

Sep 21, 2026

Single-cell transcriptomics elucidates how reactive oxygen species (ROS) signaling contributes to hepatocellular carcinoma (HCC) development. Using Seurat and CellMarker on the GSE189175 scRNA-seq dataset, pathway analyses with DAVID and MsigDB, SCENIC inference of transcription-factor networks, and CellChat for cellular communication identify six major cell subpopulations. Hepatocytes dominate in both cancer and adjacent tissues, while hallmark enrichment indicates abnormal ROS pathway activation that correlates with energy metabolism. GPX4+ hepatocytes with the highest ROS activity predict worse prognosis, with JUND positively regulating this ROS program and ligand-receptor crosstalk between GPX4+ liver cells and immune cells promoting malignancy.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages12

Reading Time30 min

AccessFree to download

Introduction

Background on HCC heterogeneity and ROS

Methods

Dataset acquisition and preprocessing

Single-cell analysis and pathway enrichment

Transcription factor network inference

Cellular communication analysis

Results

Identification of major cell subpopulations

ROS pathway activation in hepatocytes

Subgrouping and prognostic impact of GPX4+ hepatocytes

JUND regulation and immune crosstalk

Conclusion

Over-activated ROS signaling and implications for precision treatment

[www. nature.com/scientificreports](www. nature.com/scientificreports) OPEN Single-cell transcriptomics reveals over-activated reactive oxygen species pathway in hepatocytes in the development of hepatocellular carcinoma Xiaoping Wang1􀀍, Penghui Li2, Huicong Ji1, Zhenzhen Xu1 & Huiwu Xing3􀀍 Background: Hepatocellular carcinoma (HCC) is a highly heterogeneous tumor and a primary cause of cancer-relevant deaths worldwide. The role of reactive oxygen species (ROS) in HCC development is less studied. Methods: Seurat package and CellMarker database were employed for single-cell RNA sequencing (scRNA-seq) analysis based on the GSE189175 dataset from Gene Expression Omnibus (GEO). DAVID and MsigDB database were utilized for pathway analysis. SCENIC analysis was performed to map a transcription factors (TFs) regulatory network. CellChat was used for cellular communication analysis. Results: Six major cell subpopulations were identified, among which hepatocytes accounted for the highest proportion in both cancer and adjacent tissues. The enrichment scores ofthe 50 hallmark gene sets showed that the ROS pathway was abnormally activated in HCC hepatocytes and positively correlated with energy metabolism-related pathways (glucose metabolism, lipid metabolism, amino acid metabolism, etc.). Then, the hepatocytes were divided into four subgroups. Noticeably, GPX4+ hepatocytes with the highest activity ofthe ROS pathway was related to a worse prognosis of HCC. Mechanism analysis revealed that JUND was involved in the positive regulation ofthe ROS pathway in GPX4+ hepatocytes. It was found that the interdependent ligand-receptor interaction between GPX4+ liver cells and immune cells facilitated the malignant development of HCC. Conclusion: ROS pathway was over-activated in the hepatocytes of HCC tissues. GPX4+ hepatocytes having the highest activity ofthe ROS pathway closely interacted with T cells and M2 macrophage cells. Molecular subtypes and risk score signature based on the ROS pathway and its potential target gene JUND are encouraged tobe developed for improving the precision treatment of HCC. Keywords Single-cell transcriptomics, Hepatocellular carcinoma, Reactive oxygen species pathway, GPX4+ HCC hepatocytes, CellChat Abbreviations GEO GO GRN HCC PCA ROSscRNA-seqssGSEATCGA TFs UMAP Gene Expression Omnibus Gene Ontology Gene regulatory network Hepatocellular carcinoma Principal Component Analysis Reactive oxygen species Single-cell RNA sequencing Single sample gene set enrichment analysis The Cancer Genome Atlas Transcription factors Uniform Manifold Approximation and Projection 1Department of Infectious Disease, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China. 2Department of Gastrointestinal Surgery, The First Affiliated Hospital, College of Clinical Medicine, Henan University of Science and Technology, Luoyang 471000, China. 3Department of Pediatric Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China. 􀀍 [email: wangxp19731230@163.com](email: wangxp19731230@163.com); [xhwzzu@163.com](xhwzzu@163.com) [www. nature.com/scientificreports/](www. nature.com/scientificreports/) Hepatocellular carcinoma (HCC) is the most frequently detected primary liver cancer that accounts for about 90% of all liver cancer cases1,2, ranking the third highest cause for cancer-relevant deaths worldwide3. Such a high death rate is related to a late diagnosis and a high occurrence of metastasis in HCC. At present, liver transplantation, hepatectomy and ablation are the main treatments for HCC4, but 70% of patients with resectable HCC will develop intra-or extra-hepatic recurrent metastasis after surgery5,6. Moreover, a highly heterogeneous nature of HCC also significantly influences the recurrence and drug resistance in patients with HCC7. Hence, exploring the features of HCC tumor heterogeneity is instrumental for improving the clinical therapies

FAQs

What datasets and tools were used to analyze single-cell RNA sequencing in this study?

The study analyzed the GSE189175 scRNA-seq dataset from GEO using Seurat and CellMarker. Pathway analysis used DAVID and MsigDB, transcription factor regulation was mapped with SCENIC, and cellular communication was assessed with CellChat.

Which cell population showed the most abnormal ROS pathway activity, and what was its clinical significance?

GPX4+ hepatocytes exhibited the highest ROS pathway activity. This subgroup was associated with a worse prognosis in HCC.

How is the ROS pathway connected to transcriptional regulation and immune interaction?

Mechanistic analysis indicated that JUND positively regulates the ROS pathway in GPX4+ hepatocytes. Ligand-receptor interactions between GPX4+ liver cells and immune cells facilitated malignant HCC development.

Sep 21, 2026

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