【摘要】 目的 采用生物信息学方法筛选GATAD2A调控食管鳞状细胞癌(ESCC)的关键基因和通路，探讨GATAD2A在ESCC中作用的分子机制。方法 取对数生长期ESCC细胞株KYSE-150,分为敲低组(转染GATAD2A-siRNA)和对照组(转染NC-siRNA)。转染60 h取2组细胞，采用Trizol法提取总RNA,采用转录组测序(RNA-Seq)技术检测mRNA表达，应用R软件DEGseq包筛选差异表达基因，应用R软件的clusterProfiler包对差异表达基因进行基因本体(GO)功能注释分析、京都基因与基因组百科全书(KEGG)通路富集分析、基因集富集分析(GSEA)。应用STRING 12.0在线软件构建差异表达基因的蛋白互作网络(PPI),使用Cytoscape软件的MCODE插件对PPI网络进行聚类分析，筛选出得分最高的关键子网络，使用CytoHubba插件对关键子网络中的基因进行节点度计算，筛选PPI网络核心位置的节点基因，并对节点基因进行GO/KEGG分析。自TCGA数据库收集82例ESCC患者癌组织PPI分析中节点度≥10的基因表达以及预后数据，以基因表达的中位数为阈值将ESCC患者分为高、低表达组，比较高表达组与低表达组的总生存期，筛选出影响预后的基因作为关键基因。自TCGA数据库选取ESCC患者癌组织GATAD2A及关键基因的表达数据，采用Spearman法分析GATAD2A与关键基因表达的相关性。自GTEx数据库收集正常食管组织关键基因的表达数据，与ESCC组织进行比较。结果 (1)敲低组与对照组差异表达基因有888个。与对照组比较，敲低组表达上调基因275个，表达下调基因613个。(2)GO功能注释分析结果显示，前10个差异表达的基因主要参与细胞外刺激反应、细胞因子生成的正向调控、上皮细胞增殖等生物学过程。KEGG富集分析结果显示，前10个差异表达的基因主要在人乳头瘤病毒感染、PI3K/Akt信号通路、癌症相关miRNA等通路中富集。GSEA分析结果显示，差异表达基因主要在DNA合成与复制、细胞周期相关通路中富集。(3)PPI网络分析结果显示，差异表达基因共包括1 903条边缘数和486个节点基因。关键子网络包括160条边缘数和35个节点基因。GO/KEEG分析发现，节点基因主要在细胞周期调控、缺氧反应、细胞黏附等生物学过程相关。(4)PPI关键子网络筛选出13个节点度≥10的基因，Kaplan-Meier生存分析显示EGFR低表达组(EGFR表达<6.98)中位总生存期(3.99年)长于EGFR高表达组(EGFR表达≥6.98)(2.09年)(P=0.047),mTOR低表达组(mTOR表达<3.21)中位总生存期(3.73年)长于mTOR高表达组(mTOR表达≥3.21)(1.78年)(P=0.022)。ESCC组织EGFR、mTOR表达[5.30(4.47,6.38)、4.45(4.12,4.78)]均高于正常食管组织[4.44(4.14,4.70)、4.39(4.05,4.64)](U=31 720.00、52 290.00,P均<0.05)。ESCC组织GATAD2A表达为5.93(5.55,6.38),与EGFR、mTOR表达均呈正相关(r=0.295,P=0.007;r=0.398,P<0.001)。结论 GATAD2A调控ESCC的机制可能与激活EGFR和PI3K/Akt/mTOR信号通路相关。更多还原

【Abstract】 Objective To screen the key genes and pathways of esophageal squamous cell carcinoma(ESCC) regulated by GATAD2A with bioinformatics methods, and to explore the molecular mechanism of GATAD2A in ESCC. Methods The ESCC cell line KYSE-150cells in logarithmic growth phase were divided into knockdown group(transfected with GATAD2A-siRNA)and the control group(transfected with NC-siRNA).After 60hof transfection,cells were collected from both groups,and the total RNA was extracted with Trizol method.The mRNA expression was detected with RNA sequencing(RNA-Seq)technique.The differentially expressed genes were screened with R software’s DEGseq package.The Gene Ontology(GO)functional annotation analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis,and Gene Set Enrichment Analysis(GSEA)of differentially expressed genes were conducted with R software’s clusterProfiler package.The protein-protein interaction(PPI)network of differentially expressed genes was constructed with STRING 12.0online software.The cluster analysis of the PPI network was conducted with Cytoscape software’s MCODE plugin and the highest scoring key subnetwork was screened.The node degree of genes in the key subnetwork was calculated with CytoHubba plugin.The hub genes in the core positions of PPI network were screened and the GO/KEGG analysis was performed on the hub genes.The gene expression and prognostic data with a node degree≥10of PPI analysis were collected from 82ESCC patients in TCGA database.The ESCC patients were divided into the high-and low-expression groups based on the median of gene expression as threshold.The overall survival was compared between the high-and low-expression groups,and the genes affecting prognosis were screened as key genes.The expression data of GATAD2Aand key genes of cancer tissue were selected from ESCC patients in TCGA database,and the correlation between GATAD2Aand key genes expressions was analyzed with Spearman method.The expression data of key genes of normal esophageal tissues were collected from GTEx database,and were compared with ESCC tissues.Results(1)There were 888differentially expressed genes between the knockdown group and the control group.Compared with the control group,275up-regulated genes and 613down-regulated genes were expressed in the knockdown group.(2)GO functional annotation analysis results showed that the top 10differentially expressed genes were mainly involved in extracellular stimulus response,positive regulation of cytokine production,epithelial cell proliferation and other biological processes.KEGG enrichment analysis results showed that the top 10differentially expressed genes were mainly enriched in human papillomavirus infection,PI3K/Akt signaling pathway,cancer-related miRNA and other pathways.GSEA analysis results showed that the differentially expressed genes were mainly enriched in DNA synthesis and replication,and cell cycle-related pathways.(3)PPI network analysis results showed that differentially expressed genes included 1 903edges and 486hub genes.The key subnetwork included 160edges and 35hub genes.GO/KEGG analysis showed that hub genes were mainly correlated with biological processes such as cell cycle regulation,hypoxia response,and cell adhesion.(4)The PPI key subnetwork screened out 13genes with node degree≥10.Kaplan-Meier survival analysis showed that the median overall survival was longer in the EGFR low-expression group(EGFR expression<6.98)(3.99years)than that in the EGFR high-expression group(EGFRexpression≥6.98)(2.09years)(P=0.047),and was longer in the mTOR low-expression group(mTORexpression<3.21)(3.73years)than that in the mTOR high-expression group(mTORexpression≥3.21)(1.78years)(P=0.022).The expressions of EGFRand mTOR were higher in ESCC tissues[5.30(4.47,6.38),4.45(4.12,4.78)]than those in normal esophageal tissues[4.44(4.14,4.70),4.39(4.05,4.64)](U=31 720.00,52 290.00;all P values <0.05).The expression of GATAD2Ain ESCC tissues was 5.93(5.55,6.38),and was positively correlated with the expressions of EGFRand mTOR(r=0.295,P=0.007;r=0.398,P<0.001).Conclusion GATAD2A regulates ESCC probably by activating EGFRand PI3K/Akt/mTOR signaling pathways.更多还原