Integrated Analysis of Single-Cell and Bulk RNA-Sequencing Defines N7-Methylguanosine (m7G)-Mediated Modifications' Role in Prognosis and the Tumor Immune Microenvironment in Hepatocellular Carcinoma

Abstract

Background: RNA 7-Methylguanosine (m7G) modification is biologically important to tumorigenesis and progression. Knowledge regarding the effect of m7G modification on the clinical features of hepatocellular carcinoma and its prognosis, tumor immune microenvironment, and sensitivity to immunotherapy remains limited.

Method: Single-cell RNA sequencing (scRNA-seq) data was sourced from the GEO repository. Transcriptomic datasets were procured from TCGA, GEO, and ICGC databases. Following this, we scrutinized 17 m7G-associated genes and executed unsupervised clustering to discern m7G modification profiles within HCC. Evaluation of immune cell infiltration levels and associated biological functions across varied m7G modification patterns was conducted using single-sample gene set enrichment analysis (ssGSEA) complemented by gene set variation analysis (GSVA). In addition, we employed principal component analysis methodologies to elucidate m7G modification profiles in individual tumors and subsequently devised the m7Gscore. Data pertaining to m7G-related protein and mRNA levels were sourced from the Human Protein Atlas (HPA) database. These findings were corroborated in both HCC and normal samples utilizing western blot analysis, immunohistochemical assays, and RT-qPCR evaluations.

Results: Our analysis unveiled diverse cellular subgroups within HCC, with a particular focus on T-cell populations, providing insights into the intricate interplay of immune activation and suppression within the tumor microenvironment (TME). HCC patients were stratified into three distinct m7G modification patterns: immune-desert, immune-inflamed, and immune-excluded. Subsequently, m7Gscores, derived from m7G-associated signature genes, categorized HCC patients into high and low m7Gscore groups. Patients in the high-score cohort exhibited significantly improved survival outcomes compared to those in the low-score cohort. Elevated m7Gscores also correlated with enhanced therapeutic responsiveness, leading to improved outcomes in both chemotherapy and immunotherapy regimens. Our findings were further validated through RT-qPCR, western blot analyses, and immunohistochemical assays, providing robust support for our bioinformatic observations.

Conclusion: The m7G modification profiles play a pivotal role in prognosticating outcomes for HCC patients, intricately intertwined with microenvironmental heterogeneity and complexity. Furthermore, the analytical insights gained from the m7Gscore signature not only shed light on tumor microenvironment infiltration dynamics but also strengthen prognostic precision, paving the way for optimizing immunotherapeutic approaches in HCC.

Keywords: hepatocellular carcinoma microenvironment; immune checkpoint inhibitors; m7G modification; single‐cell RNA sequencing; tumor immune microenvironment.