# Stratification of Hepatocellular Carcinoma Using N6-Methyladenosine

## TL;DR

This study identifies an 8-gene signature linked to N6-methyladenosine that predicts prognosis and immunotherapy response in liver cancer patients.

## Contribution

The study introduces a novel 8-gene risk score based on m6A regulators for predicting HCC prognosis and immunotherapy outcomes.

## Key findings

- The 8-gene risk score accurately predicts HCC prognosis with AUC values over 0.75 in multiple cohorts.
- High-risk patients show reduced immune tolerance and better immunotherapy response, supported by single-cell RNA sequencing data.
- The gene ANLN promotes HCC cell proliferation and migration, as shown in in vitro experiments.

## Abstract

Hepatocellular carcinoma (HCC) is a highly heterogeneous malignant tumor. Among the various epigenetic alterations involved in its pathogenesis, the N6-methyladenosine (m6A) modification of eukaryotic mRNA plays a pivotal role in tumor biology and holds significant potential for diagnostic and therapeutic applications. In this study, we identified and validated an 8-gene risk score that robustly predicts prognosis in HCC patients, achieving area under the ROC curve (AUC) values greater than 0.75 across both internal and external cohorts. A high-risk score was significantly associated with poorer clinical outcomes and reduced immune tolerance, while also indicating a potentially enhanced response to immunotherapy. Single-cell RNA sequencing revealed a higher proportion of T cells and a decreased presence of immunosuppressive T cells in the high-risk group, which may account for their improved responsiveness to immune checkpoint inhibitors. Finally, in vitro experiments and immunohistochemical staining showed the biological function of the key gene ANLN.

Background: The N6-methyladenosine (m6A) modification of eukaryotic mRNA is the most prevalent of such epigenetic modifications and has recently been identified as a potential player in the pathogenesis and progression of hepatocellular carcinoma (HCC). With the increasing emergence of immunotherapy in the treatment of HCC, we have evaluated the potential of m6A-related genes in predicting overall survival and the therapeutic efficacy of immunotherapy in HCC patients. Methods: We employed transcriptomic data from TCGA-LIHC and GSE76427, comprising a total of 485 HCC patients, as the training set. Based on 23 recognized m6A regulators, we performed clustering analysis on HCC patients. The intersecting differentially expressed genes (DEGs) among subtypes were used in least absolute shrinkage and selection operator (LASSO) Cox and multivariate Cox regression analyses to construct the risk model. For the quantification of a risk model of HCC patients, a risk score was developed and correlated with clinical and immunological parameters. Furthermore, a single-cell transcriptomic atlas was used to analyze the relationship between model genes and immune cell subpopulations. Mechanistic studies included in vitro assays to validate the association between the m6A-related gene ANLN and the progression of HCC. Results: Internal (TCGA and GEO) and external validation (ICGC) suggested that an 8-gene risk score provides an accurate and stable prognostic assessment for HCC. Furthermore, the high-risk score, characterized by elevated TP53 mutation frequency, tumor mutation burden (TMB), and tumor stem cell characteristics indicated a poor prognosis. The prognostic signature was associated with immune cell infiltration in HCC. Those patients with a high-risk score had lower immune tolerance with a better prediction of the efficacy of immunotherapy. The risk model helps to assess and predict the response and prognosis of HCC patients to immune checkpoint inhibitors (ICIs). Additionally, single-cell RNA sequencing data revealed that the high-risk group had a higher proportion of T cells and fewer immunosuppressive T cells, potentially correlating with a better response to immunotherapy. Finally, in vitro experiments showed that ANLN, an m6A-related gene, promoted the proliferation and migration of HCC cells. Conclusions: In this study, we identified and validated an m6A gene signature consisting of eight genes that can be used to predict prognosis and immunotherapy efficacy in HCC patients.