整合转录组学识别食管鳞癌关键基因细胞周期蛋白依赖性激酶抑制因子3

Integrated Transcriptomics to Identify the Key Gene Cyclin-dependent Kinase Inhibitor 3 in Esophageal Squamous Cell Carcinoma

目的运用整合转录组学方法筛选参与食管鳞状细胞癌(ESCC)发生发展的相关基因,并观察其临床意义。方法从GEO数据库中下载ESCC表达谱的原始文件,使用R软件中的affy包读取数据并归一化,Robustrankaggreg包对各表达谱整合,以|Log2(倍数变化)|≥1.5和矫正P<0.05为标准获得差异表达基因(DEGs);利用STRING数据库构建蛋白质互作网络(PPI),cytoscape软件及MCODE插件进行网络可视化及模块挖掘,获取模块内的关键基因;采用癌症基因组图谱(TCGA)验证关键基因在ESCC组织及癌旁组织中的表达,绘制受试者工作特征(ROC)曲线,分析关键基因对ESCC的诊断价值;免疫组化检测我院184例ESCC患者癌组织标本及50例癌旁组织中关键基因的蛋白表达,分析蛋白表达与临床特征的关系。结果通过整合数据集GSE77861、GSE100942、GSE26886、GSE17351、GSE38129、GSE33426、GSE20347、GSE23400,最终获得DEGs 244个,其中上调93个、下调151个;构建PPI网络后,模块挖掘显示细胞周期蛋白依赖性激酶抑制因子3(CDKN3)被识别为模块内关键基因;TCGA验证结果显示,与癌旁组织比较,CDKN3 mRNA在ESCC组织中表达上调[3.291(IQR:2.833~3.659)vs 1.184(IQR:0.734~1.72),U=18.00,P<0.05];ROC曲线显示,曲线下面积为0.980,CDKN3 mRNA诊断ESCC的特异性为90.91%,敏感性为92.59%;免疫组化显示,ESCC组织中CDKN3的阳性表达率高于癌旁组织,差异有统计学意义(P<0.05);不同性别、年龄、T及M分期的ESCC患者CDKN3蛋白表达比较,差异无统计学意义(P>0.05);不同N分期及临床分期的ESCC患者CDKN3蛋白表达比较,差异有统计学意义(P<0.05)。结论CDKN3在ESCC组织及癌旁组织中表达存在差异,其可能是ESCC的关键基因;另外,CDKN3的表达与患者N分期及临床分期有关,其可作为诊断ESCC的生物标志物。

Objective To use integrated transcriptomics to screen the genes involved in the occurrence and development of esophageal squamous cell carcinoma(ESCC),and observe its clinical significance.Methods Download the original ESCC expression profile file from the GEO database,use the affy package in the R software to read the data and normalize it,and the Robustrankaggreg package to integrate each expression profile.Use|Log2(fold change)|≥1.5 and correction P<0.05 as the standard to obtain differentially expressed genes(DEGs);Use STRING database to construct protein interaction network(PPI),cytoscape software and MCODE plug-in for network visualization and module mining,and obtain key genes in the module;Use the Cancer Genome Atlas(TCGA)to verify the expression of key genes in ESCC tissues and adjacent tissues,draw receiver operating characteristic(ROC)curves,and analyze the diagnostic value of key genes for ESCC;Immunohistochemistry was used to detect the protein expression of key genes in cancer tissue specimens of 184 ESCC patients and 50 cases of adjacent tissues in our hospital,and analyze the relationship between protein expression and clinical features.Results By integrating data sets GSE77861,GSE100942,GSE26886,GSE17351,GSE38129,GSE33426,GSE20347,GSE23400,Finally,244 DEGs were obtained,of which 93 were up-regulated and 151 were down-regulated.After constructing the PPI network,module mining showed that cyclin-dependent kinase inhibitor 3(CDKN3)was identified as a key gene in the module;TCGA verification results showed that CDKN3 mRNA expression in ESCC tissues was up-regulated compared with adjacent tissues[3.291(IQR:2.833~3.659)vs 1.184(IQR:0.734~1.72),U=18.00,P<0.05];The ROC curve showed that the area under the curve was 0.980,the specificity of CDKN3 mRNA in diagnosing ESCC was 90.91%,and the sensitivity was 92.59%;Immunohistochemistry showed that the positive expression rate of CDKN3 in ESCC tissue was higher than that in adjacent tissues,the difference was statistically significant(P<0.05);There was no statistically significant difference in CDKN3 protein expression in ESCC patients of different gender,age,T and M stages(P>0.05);There was a statistically significant difference in the expression of CDKN3 protein in ESCC patients with different N stages and clinical stages(P<0.05).Conclusion CDKN3 is differently expressed in esophageal squamous cell carcinoma tissues and adjacent tissues,and it may be a key gene of esophageal squamous cell carcinoma;In addition,the expression of CDKN3 is related to the patient's N stage and clinical stage,and it can be used as a biomarker for clinical diagnosis of esophageal squamous cell carcinoma.