白三烯B4受体在急性髓系白血病中的预后价值和功能富集分析

Prognostic implications and functional enrichment analysis of LTB4R in patients with acute myeloid leukemia

目的运用生物信息学分析白三烯B4受体(LTB4R)在急性髓系白血病(AML)患者中的表达模式、预后价值和潜在生物学功能。方法从癌症基因组图谱(TCGA)数据库和美国国立生物技术信息中心(NCBI)公共基因芯片数据平台(GEO)下载mRNA基因芯片数据集和临床基线资料数据集,利用R studio进行表达分析、生存分析、Cox风险回归分析、相关性分析研究LTB4R的表达模式和预后价值。利用UALCAN平台研究LTB4R表达水平与临床特征的关联。利用linkedomics平台筛选出LTB4R共表达基因进行GO和KEGG信号通路富集分析。应用STRING平台构建蛋白质相互作用网络。基于LTB4R表达水平将LAML数据集分组并筛选差异表达基因,继而进行GSEA分析。收集临床30例AML患者及21例健康对照者外周静脉抗凝血2 mL,分离单个核细胞,采用qRT-PCR对LTB4R和免疫检查点基因表达水平、相关性进行验证;分离外周血500μL血清,采用酶联免疫吸附实验(ELISA)检测LTB4R蛋白表达水平。结果LTB4R表达水平在AML患者骨髓样本中显著增强(4.898±1.220 vs 2.252±0.215,P<0.001),与不良预后相关[P=0.004,风险指数(HR)=1.74],并可做为AML预后的独立风险因素(P=0.019,HR=1.66)。同时LTB4R与FAB分型、细胞遗传学风险、染色体异常、NPM1突变相关。LTB4R的共表达基因富集到多个肿瘤发生相关的信号途径,例如粒细胞炎症、淋巴细胞激活、免疫反应信号传导、能量代谢等。GSEA分析显示差异基因富集于免疫细胞分化、活化、细胞因子信号途径等。临床实验证实AML患者样本中LTB4RmRNA(P=0.044)及蛋白表达水平增高(P=0.008),并且mRNA表达水平与HAVCR2呈正相关(r=0.466,P=0.040)。结论LTB4R可做为AML的新型标志物和独立预后指标,潜在功能富集于肿瘤免疫、代谢相关信号通路,可为AML疾病进程相关信号通路和分子网络研究提供线索。

Objective To explore the expression patterns,prognostic implications,and biological role of leukotriene B4 receptor(LTB4R)in patients with acute myeloid leukemia(AML).Methods We collected the data of mRNA expression levels and clinical information of patients with AML from The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)database for mRNA expression analyses,survival analyses,Cox regression analyses and correlation analyses using R studio to assess the expression patterns and prognostic value of LTB4R.The correlation of LTB4R expression levels with clinical characteristics of the patients were analyzed using UALCAN.The co-expressed genes LTB4R were screened from Linkedomics and subjected to functional enrichment analysis.A protein-protein interaction network was constructed using STRING.GSEA analyses of the differentially expressed genes(DEGs)were performed based on datasets from TCGA-LAML stratified by LTB4R expression level.We also collected peripheral blood mononuclear cells(PBMCs)from AML patients and healthy donors for examination of the mRNA expression levels of LTB4R and immune checkpoint genes using qRT-PCR.We also examined serum LTB4R protein levels in the patients using ELISA.Results The mRNA expression level of LTB4R was significantly increased in AML patients(4.898±1.220 vs 2.252±0.215,P<0.001),and an elevated LTB4R expression level was correlated with a poor overall survival(OS)of the patients(P=0.004,HR=1.74).LTB4R was identified as an independent prognostic factor for OS(P=0.019,HR=1.66)and was associated with FAB subtypes,cytogenetic risk,karyotype abnormalities and NPM1 mutations.The co-expressed genes of LTB4R were enriched in the functional pathways closely associated with AML leukemogenesis,including neutrophil inflammation,lymphocyte activation,signal transduction,and metabolism.The DEGs were enriched in differentiation,activation of immune cells,and cytokine signaling.Examination of the clinical serum samples also demonstrated significantly increased expressions of LTB4R mRNA(P=0.044)and protein(P=0.008)in AML patients,and LTB4R mRNA expression was positively correlated with the expression of the immune checkpoint HAVCR2(r=0.466,P=0.040).Conclusion LTB4R can serve as a novel biomarker and independent prognostic indicator of AML and its expression patterns provide insights into the crosstalk of leukemogenesis signaling pathways involving tumor immunity and metabolism.