Integrated bulk and single-cell transcriptomics unveil the role of AHCY in cell cycle regulation of endometrial cancer.

Background: Although AHCY has been implicated in cancer progression, its specific role in endometrial cancer (EC) remains poorly understood. This study aimed to elucidate the oncogenic role of AHCY in EC and its association with patient prognosis. Methods: The study was initiated by analyzing AHCY expression, prognostic relevance, and immunotherapy associations across multiple cancers in TCGA Pan-Cancer project. The connection between AHCY expression and patient survival was then assessed through Kaplan-Meier survival analysis within EC datasets. Further investigations included functional annotation of AHCY, its correlation with the tumor immune microenvironment, and screening of potential targeting drugs. Single-cell RNA sequencing data were interrogated to identify key cell types associated with AHCY expression. Finally, experimental validation was conducted using clinical specimens and cell lines to examine AHCY expression, and functional consequences of AHCY knockdown-including effects on cell function, cell cycle, and apoptosis-were systematically assessed. Results: AHCY was associated with cancer prognosis, cell cycle, and response to immunotherapy, and was correlated with clinical stage and prognosis in EC. Pathways such as cell cycle and DNA replication were activated in the samples with high expression of AHCY. Most immune cells exhibit reduced infiltration in the AHCY high-expression group. Macrophages were selected as the key cell type, which exhibited a significant difference in AHCY expression between EC and control samples. The expression of AHCY was remarkably upregulated in clinical EC tissues or Ishikawa cells. AHCY knockdown exerted anti-tumor effects by concurrently impairing proliferative and metastatic capacities, enhancing apoptotic cell death, and arresting cell cycle. Folic acid, computationally identified as an AHCY-associated candidate, exerted a marked anti-proliferative effect on Ishikawa cells, demonstrating dose-dependent growth inhibition. Conclusion: AHCY is associated with patient prognosis and malignant phenotypes in EC, and macrophages represent a potentially relevant AHCY-expressing cell population within the tumor microenvironment. This study sheds light on EC pathogenesis and suggests potential for targeted therapy.