Data Sheet 4_Integrative analysis of crotonylation-associated genes reveals prognostic and therapeutic targets in gliomas.pdf

BackgroundCrotonylation, an emerging epigenetic modification, has been implicated in various biological processes, including tumor progression. However, its role in glioma remains poorly understood. This study aims to investigate the prognostic and therapeutic implications of crotonylation-associated genes in glioma.
MethodsCrotonylation levels were assessed by IHC in glioma tissues of varying grades. Key crotonylation-associated genes were identified and analyzed across five glioma datasets. A prognostic risk score was developed using machine learning algorithms and validated in multiple cohorts. Genomic alterations, immune landscapes, and therapeutic responses were examined in relation to the risk score. Single-cell dataset GSE131928 was analyzed to explore the relationship between the risk score and immune cell infiltration. After crotonate treatment of T98G cells, ChIP-seq and qPCR were performed to investigate the effect of crotonylation on gene expression. Finally, PD-1 and GZMB expression levels were assessed in glioma tissues with varying crotonylation levels.
ResultsCrotonylation levels were negatively correlated with glioma grade. Crotonylation-related genes stratified patients into two subtypes with distinct overall survival outcomes. High-risk patients exhibited increased somatic mutations, specific copy number variations, and an immunosuppressive tumor microenvironment. The risk score correlated positively with TIDE scores, indicating resistance to immune checkpoint blockade therapy. Single-cell analysis revealed a positive association between the risk score and TAM infiltration. Candidate therapeutic agents tailored for high- and low-risk groups were identified. ChIP-seq and qPCR demonstrated that reduced crotonylation suppressed CXCL1 expression and promoted GZMB expression in the glioma microenvironment.
ConclusionCrotonylation-associated genes play a pivotal role in glioma progression and prognosis. The risk score provides a robust tool for patient stratification and treatment guidance, underscoring the importance of crotonylation in glioma biology and its potential as a therapeutic target.

背景：巴豆酰化（Crotonylation）作为一类新兴的表观遗传修饰，已被证实参与包括肿瘤进展在内的多种生物学进程。然而其在胶质瘤中的作用仍有待阐明。本研究旨在探究巴豆酰化相关基因在胶质瘤中的预后及治疗价值。
方法：采用免疫组化（Immunohistochemistry, IHC）检测不同级别胶质瘤组织的巴豆酰化水平。通过分析5个胶质瘤数据集，筛选并鉴定关键巴豆酰化相关基因。利用机器学习算法构建预后风险评分模型，并在多队列中进行验证。结合风险评分，分析基因组变异、免疫微环境特征及治疗响应情况。采用单细胞数据集GSE131928，探究风险评分与免疫细胞浸润的关联。对T98G细胞进行巴豆酸盐处理后，通过染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）与实时定量聚合酶链反应（Quantitative Polymerase Chain Reaction, qPCR），探究巴豆酰化对基因表达的调控作用。最后，检测不同巴豆酰化水平胶质瘤组织中程序性死亡受体1（Programmed Death 1, PD-1）与颗粒酶B（Granzyme B, GZMB）的表达水平。
结果：巴豆酰化水平与胶质瘤级别呈负相关。巴豆酰化相关基因可将患者分为两个亚型，二者总生存期存在显著差异。高风险组患者表现出更高的体细胞突变负荷、特异性拷贝数变异以及免疫抑制性肿瘤微环境。风险评分与肿瘤免疫功能异常和排斥评分（Tumor Immune Dysfunction and Exclusion, TIDE）呈正相关，提示高风险患者对免疫检查点阻断治疗存在耐药性。单细胞分析显示，风险评分与肿瘤相关巨噬细胞（Tumor-associated macrophages, TAM）浸润呈正相关。本研究筛选出针对高低风险组的潜在治疗药物。ChIP-seq与qPCR实验证实，巴豆酰化水平降低可抑制胶质瘤微环境中CXCL1的表达，并促进GZMB的表达。
结论：巴豆酰化相关基因在胶质瘤进展与预后中发挥关键作用。本研究构建的风险评分模型可实现有效的患者分层与治疗指导，凸显了巴豆酰化在胶质瘤生物学中的重要性及其作为治疗靶点的潜在价值。