Subtype cluster analysis unveiled the correlation between m6A- and cuproptosis-related lncRNAs and the prognosis, immune microenvironment, and treatment sensitivity of esophageal cancer
Authors: Zhang Ming , Su Yani , Wen Pengfei , Shao Xiaolong , Yang Peng , An Peng , Jing Wensen , Liu Lin , Yang Zhi , Yang Mingyi 
Published: February 2025

Objective

Esophageal cancer (EC) is characterized by a high degree of malignancy and poor prognosis. N6-methyladenosine (m6A), a prominent post-transcriptional modification of mRNA in mammalian cells, plays a pivotal role in regulating various cellular and biological processes. Similarly, cuproptosis has garnered attention for its potential implications in cancer biology. This study seeks to elucidate the impact of m6A- and cuproptosis-related long non-coding RNAs (m6aCRLncs) on the prognosis of patients with EC.

Methods

The EC transcriptional data and corresponding clinical information were retrieved from The Cancer Genome Atlas (TCGA) database, comprising 11 normal samples and 159 EC samples. Data on 23 m6A regulators and 25 cuproptosis-related genes were sourced from the latest literature. The m6aCRLncs linked to EC were identified through co-expression analysis. Differentially expressed m6aCRLncs associated with EC prognosis were screened using the limma package in R and univariate Cox regression analysis. Subtype clustering was performed to classify EC patients, enabling the investigation of differences in clinical outcomes and immune microenvironment across patient clusters. A risk prognostic model was constructed using least absolute shrinkage and selection operator (LASSO) regression. Its robustness was evaluated through survival analysis, risk stratification curves, and receiver operating characteristic (ROC) curves. Additionally, the model’s applicability across various clinical features and molecular subtypes of EC patients was assessed. To further explore the model’s utility in predicting the immune microenvironment, single-sample gene set enrichment analysis (ssGSEA), immune cell infiltration analysis, and immune checkpoint differential expression analysis were conducted. Drug sensitivity analysis was performed to identify potential therapeutic agents for EC. Finally, the mRNA expression levels of m6aCRLncs in EC cell lines were validated using reverse transcription quantitative polymerase chain reaction (RT-qPCR).

Results

We developed a prognostic risk model based on five m6aCRLncs, namely ELF3-AS1, HNF1A-AS1, LINC00942, LINC01389, and MIR181A2HG, to predict survival outcomes and characterize the immune microenvironment in EC patients. Analysis of molecular subtypes and clinical features revealed significant differences in cluster distribution, disease stage, and N stage between high- and low-risk groups. Immune profiling further identified distinct immune cell populations and functional pathways associated with risk scores, including positive correlations with naive B cells, resting CD4+ T cells, and plasma cells, and negative correlations with macrophages M0 and M1. Additionally, we identified key immune checkpoint-related genes with significant differential expression between risk groups, including TNFRSF14, TNFSF15, TNFRSF18, LGALS9, CD44, HHLA2, and CD40. Furthermore, nine candidate drugs with potential therapeutic efficacy in EC were identified: Bleomycin, Cisplatin, Cyclopamine, PLX4720, Erlotinib, Gefitinib, RO.3306, XMD8.85, and WH.4.023. Finally, RT-qPCR validation of the mRNA expression levels of m6aCRLncs in EC cell lines demonstrated that ELF3-AS1 expression was significantly upregulated in the EC cell lines KYSE-30 and KYSE-180 compared to normal esophageal epithelial cells.

Conclusion

This study elucidates the role of m6aCRLncs in shaping the prognostic outcomes and immune microenvironment of EC. Furthermore, it identifies potential therapeutic agents with efficacy against EC. These findings hold significant promise for enhancing the survival of EC patients and provide valuable insights to inform clinical decision-making in the management of this disease.