成人与儿童急性髓系白血病患者肿瘤免疫相关的差异表达基因分析

doi:10.3969/j.issn.1674-8115.2021.05.004

摘要/Abstract

摘要：

目的·分析成人与儿童急性髓系白血病（acute myeloid leukemia，AML）患者骨髓中肿瘤免疫相关的差异表达基因（differentially expressed genes，DEGs）。方法·从GEO（Gene Expression Omnibus）数据库下载GSE134589数据集，选取初次确诊/复发状态的患者，按年龄分为儿童组（0~16岁，34例）、中青年组（17~59岁，62例）和老年组（60~80岁，62例），用R语言程序包筛选不同组患者骨髓样本中肿瘤免疫相关的DEGs。选取中青年组与儿童组，老年组与儿童组共同的DEGs，与完全缓解状态的成人与儿童患者的DEGs进行比对，并进行功能富集分析。利用Kaplan-Meier法筛选与预后显著相关的基因，通过构建蛋白质相互作用（protein-protein interaction，PPI）网络筛选核心调控基因，将以上2种方法筛选出的基因视为关键基因。用GEPIA服务器对比关键基因在AML、弥漫大B细胞淋巴瘤（diffuse large B cell lymphoma，DLBCL）和胸腺癌的肿瘤样本与正常人样本的表达量，在GEXC网站分析关键基因在AML患者肿瘤干细胞和健康人的原始造血细胞中mRNA的表达情况。结果·GSE134589数据集中，中青年组与儿童组比，上调的DEGs有51个，下调的有21个；老年组与儿童组比，上调的DEGs有47个，下调的有20个；而中青年组与老年组比，没有发现DEG。筛选了中青年组和老年组共同的肿瘤免疫相关DEGs 57个，其中上调有39个，下调有18个，仅有3个基因的表达水平差异在疾病完全缓解时仍有统计学意义。57个共同DEGs主要富集在白细胞迁移和细胞因子介导的信号通路，其中白细胞介素2受体α亚基（interleukin-2 receptor subunit alpha，IL2RA）、FMS-样酪氨酸激酶3（FMS-like tyrosine kinase 3，FLT3）高表达的患者与低表达的患者相比总体生存期显著缩短（均P<0.05），补体成分3a受体1（complement component 3a receptor 1，C3AR1）是PPI网络的核心调控基因。这3个基因作为关键基因，均在AML肿瘤样本特异性高表达（均P<0.05），IL2RA还在DLBCL患者样本中显著高表达（P<0.05）。IL2RA在AML肿瘤干细胞和健康人的原始造血细胞中均低表达，FLT3均高表达，而C3AR1的表达在AML肿瘤干细胞特异性升高。结论·成人与儿童AML患者预后的差异可能与骨髓中肿瘤免疫相关基因表达的差异有关，其中IL2RA、FLT3和C3AR1可能是发挥重要作用的关键基因。

关键词: 急性髓系白血病, 肿瘤免疫, 差异表达基因, 白细胞介素2受体α亚基, FMS-样酪氨酸激酶3, 补体成分3a受体1

Abstract:

Objective·To analyze tumor immune-related differentially expressed genes (DEGs) in the bone marrow of adults and children with acute myeloid leukemia (AML).

Methods·The GSE134589 data set was downloaded from the GEO (Gene Expression Omnibus) database, and the patients in initial diagnosis/relapse were selected and divided into children group (0?16 years old, 34 cases), young and middle-aged group (17?59 years old, 62 cases) and elders group (60?80 years old, 62 cases). The tumor immune-related DEGs in the bone marrow samples of different groups of patients were screened by R language packages. The common DEGs of young and middle-aged group vs children group and elders group vs children group were compared with the DEGs of adults group vs children group in complete remission, and the functional enrichment analysis was conducted with the common DEGs. The Kaplan-Meier method was used to screen the DEGs that were significantly related to prognosis, and the core regulatory DEGs were screened by constructing a protein-protein interaction (PPI) network. The genes screened by the above two methods were regarded as key genes. The expression levels of the key genes in AML, diffuse large B cell lymphoma (DLBCL) and thymoma tumor samples and normal human samples were compared by GEPIA server, and the mRNA expression levels of key genes in the human AML tumor stem cells and the original hematopoietic cells of healthy people were analyzed by GEXC website.

Results·In the GSE134589 data set, there were 51 DEGs up-regulated and 21 down-regulated between the young and middle-aged group and the children group; 47 DEGs were up-regulated and 20 down-regulated between the elders group and the children group; no DEG was found between the young and middle-aged group and the elders group. Fifty-seven tumor immune-related DEGs were screened in both the young and middle-aged group and the elders group, of which 39 were up-regulated and 18 were down-regulated; in these DEGs only 3 genes showed statistically significant difference in expression level when the disease was incomplete remission. The 57 common DEGs were mainly enriched in leukocyte migration and cytokine-related signal pathways. The patients with high expression of interleukin-2 receptor subunit alpha (IL2RA) and FMS-like tyrosine kinase 3 (FLT3) had significantly shorter overall survival than those with low expression (both P<0.05), and complement component 3a receptor 1 (C3AR1) was the core regulatory gene of the PPI network. The three DEGs were selected as key genes. They were all specifically highly expressed in the AML tumor samples (all P<0.05), and IL2RA was also significantly highly expressed in the DLBCL samples (P<0.05). The expression level of IL2RA was low both in the AML tumor stem cells and the original group of hematopoietic cells, but FLT3 was highly expressed in these cells. The expression level of C3AR1 was specifically high in the AML tumor stem cells.

Conclusion·The difference in the prognosis between adults and children with AML may be related to the differences in the expression of tumor immune-related genes in bone marrow, in which IL2RA, FLT3 and C3AR1 may be the key genes.

Key words: acute myeloid leukemia (AML), tumor immunity, differentially expressed gene (DEG), interleukin-2 receptor subunit alpha (IL2RA), FMS-like tyrosine kinase 3 (FLT3), complement component 3a receptor 1 (C3AR1)

中图分类号:

R733.71

李玲玲, 李倩, 李明玉, 刘峥, 沈倩诚. 成人与儿童急性髓系白血病患者肿瘤免疫相关的差异表达基因分析[J]. 上海交通大学学报(医学版), 2021, 41(5): 579-587.

Ling-ling LI, Qian LI, Ming-yu LI, Zheng LIU, Qian-cheng SHEN. Analysis of tumor immune-related differentially expressed genes in adults and children with acute myeloid leukemia[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(5): 579-587.

图/表 7

图1 不同年龄分组的AML患者肿瘤免疫相关基因表达的热图分析Note: A. The mRNA expression heatmap of immune genes in the young and middle-aged group and the children group. B. The mRNA expression heatmap of immune genes in the elders group and the children group. Orange and red representing the up-regulated genes, blue representing the down-regulated genes, and shades of the color representing the degrees of change in the expression.

Fig 1 Heatmap analysis of tumor immune-related genes expression in the AML patients of different age groups

图2 不同年龄分组的AML患者肿瘤免疫相关基因的火山图分析Note: A. DEG analysis of the young and middle-aged group and the children group. B. DEG analysis of the elders group and the children group. C. DEG analysis of the young and middle-aged group and the elders group.

Fig 2 Volcano map analysis of tumor immune-related genes in the AML patients of different age groups

图3 不同年龄分组的AML患者肿瘤免疫相关DEGs的韦恩图与富集分析Note: A. Thirty-nine up-regulated DEGs in both the elders group and the young and middle-aged group when compared to the children group. B. Up-regulated DEGs (n=39) mapped to the GO, Reactome and KEGG biological processes and pathways. C. Eighteen down-regulated DEGs in both the elders group and the young and middle-aged group when compared to the children group. D. Down-regulated DEGs (n=18) mapped to the GO, Reactome and KEGG biological processes and pathways.

Fig 3 Venn map and enrichment analysis of tumor immune-related DEGs in the AML patients of different age groups

表1 AML患者在初次确诊/复发和完全缓解状态下的肿瘤免疫相关DEGs的比较

Tab 1 Comparison of tumor immune-related DEGs between the AML patients in initial diagnosis/relapse and in complete remission

Disease status	Up-regulated DEG	Down-regulated DEG
Initial diagnosis/relapse^①	CD63, *CD53, CD44, CD58, CD74, CD99, CD97, CD244, CD27, CD22, CD3E, CD200, MAPK14, IL1RAP, CASP1, REL, C3AR1, FLT3, RUNX1, BCL2L1, TNFAIP3, TLR1, TNFSF13B, ITGA2B, CD275, PDGFC, TPSAB1, IL2RA, ABCB1, IL32, CCL25, CXCL16, CCL2, CCL23, CXCL2, CCL4, CCL1, CXCL12, KIT*	CD55, MAPK11, CCL24, SPACA3, CD79A, CASP10, CD40LG, CD6, IL34, C3, IL1RAPL2, CD290, CD274, CD9, CD49b, CXCL13, LCN2, LTF
Complete remission^②	IL8, CD83, CD87, *REL*, CDKN1A, *CXCL2*, CD141, CD54, CD354, *CD53*	BLNK, IKBKB

图4 IL2RA和FLT3 mRNA不同表达水平的AML患者的总体生存期分析Note: A. Overall survival analysis of IL2RA mRNA in the two groups. B. Overall survival analysis of FLT3 mRNA in the two groups.

Fig 4 Overall survival analysis of the AML patients with different levels of IL2RA and FLT3 mRNA expression

图5 上调DEGs对应蛋白的PPI网络与最显著模块图Note: A. PPI network of the 39 up-regulated DEGs. B. Results of EPC algorithm provided by CytoHubba to get the top 18 node proteins network. The shade of the node color indicating its score ranking, and the darker red representing the higher score. C. The most significant module in the PPI network was the protein network centered on C3RA1.

Fig 5 PPI network and the most significant modules of up-regulated DEGs-corresponding proteins

图6 关键基因在3个病种肿瘤组织中以及在AML患者肿瘤干细胞与正常人造血细胞中的mRNA表达情况Note: A. IL2RA, FLT3 and C3AR1 mRNA expression in the tumor tissues of AML, DLBC, and THYM and the normal samples. T—tumor; N—normal. *P<0.05. B. IL2RA, FLT3 and C3AR1 mRNA expression in the original group of hematopoietic cells in the normal human bone marrow and the umbilical cord blood as well as in the AML patients. On the left, probeset meta profile 25 229 microarrays in GEO was analyzed by Robust Multi-chip Average (RMA) and the StepMiner algorithm, and the distribution of expression levels is displayed by a histogram. Yellow represents active expression and purple represents inactive expression. CB—umbilical cord blood; HSC—hematopoietic stem cell; MPP—multipotent progenitor; RA-pos—CD45RA positive cell; BM—bone marrow; CMP—common myeloid progenitor; MEP—megakaryocyte-erythrocyte progenitor; GMP—granulocyte-monocyte progenitor; LSC—leukemia stem cell; LPC—leukemic progenitor cell; Blast—blast cell.

Fig 6 mRNA expression of key genes in the tumor tissues of the three diseases and in the tumor stem cells of the AML patients and the hematopoietic cells of normal people

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