结直肠癌（CRC）是一个重大的全球健康挑战，其发病率和死亡率不断上升。尽管免疫疗法取得了进步，但耐药性，特别是由于 T 细胞耗竭引起的耐药性，仍然是一个主要障碍。本研究探讨了 N4-乙酰胞苷 (ac4C) 修饰介导的 YWHAH 在 CRC 进展中的作用及其对 CD8 T 细胞耗竭的影响。使用乙酰化 RNA 免疫沉淀和测序 (acRIP-seq) 对五对 CRC 患者组织样本进行分析，鉴定出 ac4C 修饰的 mRNA。功能测定，包括细胞培养、转染、qRT-PCR 和免疫测定，研究了 YWHAH 表达对 CRC 进展的影响。 TCGA 数据的生物信息学分析评估了 YWHAH 与免疫反应以及检查点抑制剂之间的相关性。流式细胞术和免疫组织化学验证了这些发现，并辅以涉及 CD8 T 细胞和 CRC 细胞系（LOVO 和 HCT116）的共培养实验。 acRIP-seq 揭示 YWHAH 是 CRC 进展的潜在驱动因素，表现出 ac4C 修饰介导的稳定性和上调。高 YWHAH 水平与 CRC 患者的不良后果和免疫逃避相关，与免疫检查点蛋白有很强的相关性，与 CD8 T 细胞浸润有一定的相关性。共培养实验证明 YWHAH 诱导 CD8 T 细胞耗竭，其特征是增殖减少和耗竭标记物增加。 NAT10 介导的 ac4C 修饰增强了 YWHAH 在 CRC 中的稳定性。 YWHAH 参与 CD8 T 细胞耗竭表明其作为 CRC 免疫治疗中的治疗靶点和预后标记物的潜力，强调了 CRC 进展中表观转录组修饰、肿瘤微环境和免疫反应之间复杂的相互作用。版权所有 © 2024。由 Elsevier B.V. 出版。

Colorectal cancer (CRC) is a significant global health challenge, with increasing rates of incidence and mortality. Despite advancements in immunotherapy, resistance, particularly due to T cell exhaustion, remains a major hurdle. This study explores the role of YWHAH, mediated by N4-acetylcytidine (ac4C) modification, in CRC progression and its impact on CD8+ T cell exhaustion. Analysis of five paired CRC patient tissue samples using acetylated RNA immunoprecipitation and sequencing (acRIP-seq)identified ac4C-modified mRNAs. Functional assays, including cell culture, transfection, qRT-PCR, and immune assays, investigated the influence of YWHAH expression on CRC advancement. Bioinformatics analysis of TCGA data assessed the correlation between YWHAH and immune responses, as well as checkpoint inhibitors. Flow cytometry and Immunohistochemistry validated these findings, complemented by a co-culture experiment involving CD8+ T cells and CRC cell lines (LOVO and HCT116). acRIP-seq revealed YWHAH as a potential driver of CRC progression, exhibiting ac4C modification-mediated stability and upregulation. High YWHAH levels correlated with adverse outcomes and immune evasion in CRC patients, showing strong associations with immune checkpoint proteins and modest correlations with CD8+ T cell infiltration. Co-culture experiments demonstrated YWHAH-induced CD8+ T cell exhaustion, characterized by decreased proliferation and increased exhaustion markers. NAT10-mediated ac4C modification enhanced YWHAH stability in CRC. The involvement of YWHAH in CD8 + T cell exhaustion suggests its potential as a therapeutic target and prognostic marker in CRC immunotherapy, highlighting the intricate interplay between epitranscriptomic modifications, the tumor microenvironment, and immune responses in CRC progression.Copyright © 2024. Published by Elsevier B.V.