Construction of a Prognostic Model and Subgroup Characteristics Related to Hypoxia-Immune Evasion in T Cells of Hepatocellular Carcinoma Based on Single-Cell and Bulk RNA Analysis

Abstract: T cells, hypoxia and immune escape have complex relationships in hepatocellular carcinoma (HCC). However, the prognostic value of T cell hypoxia and immune escape-related differentially expressed genes (T-HIERDEGs) remains unknown. This study aims to explore the prognostic value of T-HIERDEGs in HCC under hypoxic microenvironment and to construct a corresponding predictive model. We integrated public single-cell and batch transcriptome data, combined with hypoxia and immune escape gene sets, to identify T cell-related hypoxia-immune escape genes. A prognostic risk model consisting of 13 genes was established and validated in the TCGA cohort, and it showed good predictive performance in multiple independent GEO cohorts. Further analysis revealed significant differences in immune microenvironment, mutation burden, and drug sensitivity among patients in different risk groups. Single-cell analysis identified four T cell subpopulations, among which the TIGIT+ and ANXA1+ subpopulations had higher characteristic scores and were closely related to hypoxia, metabolism, and immune regulatory pathways. The model genes showed high specificity of expression in these subpopulations. In vitro experiments further verified the expression of model genes in HCC cell lines. The results of functional experiments showed that knockdown of LGALS3 could inhibit cell proliferation and invasion and promote apoptosis, while affecting drug sensitivity to Dasatinib. In summary, this study constructed a robust prognostic model based on T-HIERDEGs, systematically revealed the immune and molecular characteristics of different risk groups, explained the heterogeneity of T cell subpopulations in the hypoxic microenvironment and their potential role in immune escape, and provided a new theoretical basis and candidate targets for the prognosis assessment, immunotherapy, and combined strategies of HCC. Keywords: hepatocellular carcinoma, T cells, hypoxia, immune escape, single-cell and bulk RNA-sequencing, prognostic model