慢性髓系白血病干细胞核心基因及关键通路的生物信息学分析和初步临床验证

Bioinformatics analysis and preliminary clinical validation of core genes and key pathways in chronic myeloid leukemia stem cells

目的:明确慢性髓系白血病干细胞(CML-LSC)中关键基因模块和信号通路。方法:筛选基因表达综合数据库(GEO)中包含CML-LSC和正常造血干细胞(HSC)对照组的数据集,获得GSE5550、GSE43754、GSE479273个基因芯片数据集。以|log_(2)差异倍数(FC)|>1以及P<0.05为标准筛选数据库中的差异表达基因(DEGs)。对DEGs进行通路和基因功能富集,并构建蛋白质相互作用网络(PPI),分析关键基因簇及核心基因,并对核心基因进行受试者工作特征(ROC)曲线绘制,筛选出潜在基因靶点,进行基因集富集(GSEA)分析。应用实时荧光定量PCR(RT-qPCR)和免疫荧光染色检测其在CML慢性期和急变期临床样本中的表达差异。结果:共获得52个DEGs,其中,表达升高14个,表达下调38个。这些DEGs与细胞-细胞接触区、对金属离子的反应、钙依赖的细胞-细胞通过质膜细胞黏附分子、质膜脂筏、多肽结合、Toll样受体结合、生长因子结合等细胞生物学过程相关。京都基因与基因组百科全书(KEGG)分析富集于肿瘤的转录失调信号这一重要通路,构建PPI网络,筛选出3个功能模块,并获得13个与DNA复制检查点等有关的核心基因。通过对核心基因ROC曲线绘制,选出7个在CML-LSC中有显著差异且对该疾病模型具有较好诊断价值的核心关键基因。对FMS样酪氨酸激酶3(FLT3)进行GSEA分析和重要通路的可视化(如剪接体,错配修复,碱基切除修复,DNA复制、神经活性配体受体相互作用通路)。而RT-qPCR和免疫荧光染色结果均显示FLT3在CML急变期LSC中mRNA和蛋白表达水平低于慢性期(P<0.0001)。结论:通过筛选CML-LSC中的核心基因及关键信号通路,发现转录失调信号通路对CML急变起着关键作用,而FLT3基因失调可能是导致CML疾病进展的关键基因,这为CML急变的监测和防治提供了新的潜在分子靶点。

Objective To identify key gene modules and signaling pathways in chronic myeloid leukemia stem cells(CML-LSC).Methods Three gene chip datasets,GSE5550,GSE43754 and GSE47927 were selected from Gene Expression Omnibus(GEO)database,which included CML-LSC and normal hematopoietic stem cell(HSC)control group.The differentially expressed genes(DEGs)were screened using|log_(2) fold change(FC)|>1 and P<0.05.Pathway and gene function enrichment were performed for DEGs,protein interaction network(PPI)was constructed,key gene clusters and core genes were analyzed,receiver operator characteristic curve(ROC)curves were drawn for core genes,potential gene targets were screened,and gene set enrichment(GSEA)analysis was performed.Real time fluorescence quantitative PCR(RT-qPCR)and immunofluorescence staining were used to detect the expression difference between chronic and acute CML clinical samples.Results A total of 52 DEGs were obtained,of which 14 were increased in expression and 38 were decreased in expression.These DEGs were associated with cell biological processes such as cell-cell contact regions,response to metal ions,calcium-dependent cell-cell passage through plasma membrane cell adhesion molecules,plasma membrane lipid rafts,peptide binding,Toll-like receptor binding,and growth factor binding.The Kyoto Encyclopedia of Genes and Genomes(KEGG)analyzed this important pathway of transcriptional dysregulation signals enriched in tumors.PPI network was constructed,3 functional modules were screened,and 13 core genes related to DNA replication checkpoints were obtained.Through ROC curve mapping of core genes,7 core key genes with significant differences in CML-LSC and good diagnostic value for the disease model were selected,and Fms-like tyrosine kinase 3(FLT3)played an important role in these genes.GSEA analysis of FLT3 and visualization of important pathways(such as splicosome,mismatch repair,base excise repair,DNA replication,neuroactive ligand receptor interaction pathway).RT-qPCR and immunofluorescence staining results showed that the mRNA and protein expression levels of FLT3 in the acute phase of CML-LSC were significantly lower than those in thechronic phase (P < 0.000 1). Conclusions By screening the core genes and key signaling pathways in CML-LSC, it is found thatthe transcriptional dysregulation signaling pathway plays a key role in CML rapid change, and the dysregulation of FLT3 gene maybe the key gene leading to the progression of CML disease, which provides a new potential molecular target for the monitoring andprevention of CML rapid change.