DataSheet1_Comprehensive analysis of the prognosis and immune infiltrates for the BET protein family reveals the significance of BRD4 in glioblastoma multiforme.ZIP

Background: Glioblastoma multiforme (GBM) is the most common and invasive primary central nervous system tumor. The prognosis after surgery, radiation and chemotherapy is very poor. Bromodomain (BRD) proteins have been identified in oncogenic rearrangements, and play a key role in the development of multiple cancers. However, the relationship between BET proteins and prognosis of GBM are still worth exploring, and the distinct functions of BET proteins and tumor immunology in GBM have not been fully elucidated. Therefore, it is particularly important to develop effective biomarkers to predict the prognosis of GBM patients.
Methods: Metascape, David, Kaplan-Meier Plotter, Oncomine, GEPIA, TCGA, TIMER, and LinkedOmics databases were used to assess the expression and prognosis for BET proteins in GBM. ROC analysis of risk model was established to identify the correlation between BET genes and overall survival in GBM patients. TIMER and GEPIA databases were used to comprehensively investigate the correlation between BET genes and tumor immune infiltration cells. Moreover, the image of immunohistochemistry staining of BET proteins in normal tissue and tumor tissue were retrived from the HPA database. In addition, differential analysis and pathway enrichment analysis of BRD4 gene expression profile were also carried out. Finally, immune-fluorescence and Western blot were used to clarify the expression of BRD4 in GBM cells.
Results: Bioinformatics analysis showed that the expression levels of BET genes in GBM may play an important role in oncogenesis. Specifically, bioinformatic and immunohistochemistry analysis showed that BRD4 protein was more highly expressed in tumor tissues than that in normal tissues. The high expression of BRD4 was associated with poor prognosis in GBM. The expression of BET genes were closely related to the immune checkpoint in GBM. The correlation effect of BRD4 was significantly higher than other BET genes, which represented negative correlation with immune checkpoint. The expression of BRD4 was positively associated with tumor purity, and negatively associated with immune infiltration abundance of macrophage, neutrophil and CD8+ T-cell, respectively. Cox analysis showed that the model had good survival prediction and prognosis discrimination ability. In addition, the expression levels of BRD4 protein was significantly higher in U-251 MG cells than that in normal cells, which was consistent with the results of bioinformatics data.
Conclusion: This study implied that BRD4 could be hopeful prognostic biomarker in GBM. The increased expression of BRD4 may act as a molecular marker to identify GBM patients with high-risk subgroups. BRD4 may be a valuable prognostic biomarker, and a potential target of precision therapy against GBM.

背景：多形性胶质母细胞瘤（Glioblastoma multiforme, GBM）是最常见且侵袭性最强的原发性中枢神经系统肿瘤。患者接受手术、放疗及联合化疗后的预后极差。溴结构域（Bromodomain, BRD）蛋白已在致癌重排事件中被鉴定出，并在多种癌症的发生发展中发挥关键作用。然而，BET蛋白（Bromodomain and Extra-Terminal domain, BET）与GBM预后的关联仍有待深入探索，BET蛋白在GBM中的独特功能及其与肿瘤免疫学的相互作用尚未完全阐明。因此，开发可预测GBM患者预后的有效生物标志物显得尤为重要。
方法：本研究采用Metascape、David、Kaplan-Meier Plotter、Oncomine、GEPIA、TCGA、TIMER及LinkedOmics数据库，评估GBM中BET蛋白的表达水平与预后情况。通过构建风险模型的ROC分析，明确BET基因与GBM患者总生存期的相关性。利用TIMER与GEPIA数据库，全面探究BET基因与肿瘤免疫浸润细胞的关联。此外，从人类蛋白质图谱（Human Protein Atlas, HPA）数据库中检索BET蛋白在正常组织与肿瘤组织中的免疫组化染色图像。同时，对BRD4基因的表达谱进行差异分析及通路富集分析。最后，采用免疫荧光技术与蛋白质印迹（Western blot）明确BRD4在GBM细胞中的表达水平。
结果：生物信息学分析显示，BET基因在GBM中的表达水平可能在肿瘤发生过程中发挥重要作用。具体而言，生物信息学与免疫组化分析表明，BRD4蛋白在肿瘤组织中的表达量显著高于正常组织。BRD4高表达与GBM患者的不良预后密切相关。BET基因的表达与GBM中的免疫检查点存在紧密关联，其中BRD4的相关效应显著高于其他BET基因，且与免疫检查点呈负相关。BRD4的表达与肿瘤纯度呈正相关，分别与巨噬细胞、中性粒细胞及CD8+ T细胞的免疫浸润丰度呈负相关。Cox回归分析显示，该模型具备良好的生存预测与预后区分能力。此外，U-251 MG细胞中BRD4蛋白的表达水平显著高于正常细胞，这与生物信息学数据的分析结果一致。
结论：本研究提示BRD4有望成为GBM的潜在预后生物标志物。BRD4表达升高可作为分子标志物，用以识别高风险亚群的GBM患者。BRD4可能是一种具有临床价值的预后生物标志物，同时也是GBM精准治疗的潜在靶点。