应用生物信息学分析筛选肝细胞癌关键基因及表达验证

Screening and expression verification of key genes in hepatocellular carcinoma by bioinformatics analysis

目的通过对肝细胞癌关键基因的筛选从而探讨其在肝细胞癌中的临床意义及可能的潜在机制。方法从GEO数据库中获取肝细胞癌基因芯片,通过GEO2R在线工具及Venn图筛选差异表达基因(differentially expressed genes,DEGs),利用生物学信息注释数据库(DAVID)进行GO分析和KEGG通路分析,应用STRING及Cytscape软件筛选出核心基因,将核心基因通过Kaplan-Meier Plotter数据库进行生存分析,再通过GEPIA数据库进行表达量分析,将与预后相关且在肝细胞癌中高表达的核心基因经基因富集分析在线工具Metascape进行功能及通路富集分析,最后将关键基因在所选取的临床病例的肝细胞癌及其癌旁组织中进行验证。结果从GEO数据库获取了符合要求的3个基因芯片GSE14520、GSE60502和GSE102079,共筛选出DEGs94个。对筛选出的DEGs通过上述相应分析后,筛选出19个核心DEGs,将19个核心DEGs通过Kaplan-Meier Plotter生存分析后,发现有9个基因即核糖核苷酸还原酶调节亚基M2(RRM2)、多梳抑制复合物1(PRC1)、DNA拓扑异构酶Ⅱα(TOP2A)、极光激酶A(AURKA)、核仁和纺锤体相关蛋白1(NUSAP1)、Rac-GTPase激活蛋白1(RACGAP1)、纺锤体微管组装因子(ASPM)、细胞周期蛋白依赖性激酶1(CDK1)和GINS复合体1(GINS1)与肝细胞癌的预后相关(P<0.05)。将这9个基因通过GEPIA数据库进行表达量分析,结果9个基因均在肝细胞癌组织中高表达(P<0.05)。通过Metascape在线分析工具将9个高表达基因进行功能及通路富集分析。选取RRM2在肝细胞癌组织和癌旁组织中进行验证,发现RRM2在肝细胞癌组织中的染色评分为(10.9±1.5)分,明显高于其在癌旁组织中的染色评分[(4.5±1.2)分],P<0.001。结论通过生物信息学方法分析得出的9个基因可能是肝细胞癌发生发展的关键基因,可为进一步研究肝细胞癌的发生机制、诊断及治疗提供参考。

Objective To explore the clinical significance and possible potential mechanism of hepatocellular carcinoma through the screening of key genes in hepatocellular carcinoma.Methods Hepatocellular carcinoma gene chip was obtained from GEO database,differentially expressed genes(DEGs)were screened by GEO2R online tools and Venn map,GO analysis and KEGG pathway analysis were performed in DAVID database,core genes were screened by STRING and Cytscape software,core genes were analyzed in Kaplan-Meier Plotter for survival analysis,and expression was analyzed by GEPIA database.The core genes related to prognosis and highly expressed in hepatocellular carcinoma were analyzed by Metascape online tool for function and pathway enrichment analysis.Finally,the key genes were verified in hepatocellular carcinoma and paracancerous tissues.Results A total of 94 DEGs were screened from three gene chips GSE14520,GSE60502,and GSE102079,obtained from GEO.After the selected DEGs was analyzed by GO function analysis,KEGG pathway enrichment analysis,STRING and Cytscape software by DAVID,19 core DEGs were screened.After 19 core DEGs were analyzed by Kaplan-Meier Plotter website,9 genes[ribonucleotide reductase M2(RRM2),polycomb repressive complex 1(PRC1),topoisomeraseⅡalpha(TOP2 A),aurora kinase A(AURKA),nucleolar spindleassociated protein 1(NUSAP1),Rac-GTPase activating protein 1(RACGAP1),abnormal spindle-like microcephalyassociated(ASPM),cyclin dependent kinase 1(CDK1)and GINS complex subunit 1(GINS1)]were found to be associated with the prognosis of hepatocellular carcinoma.The expressions of these 9 genes were analyzed by GEPIA,and the results showed that all 9 genes were highly expressed in hepatocellular carcinoma tissues.The functions and pathways of 9 highly expressed genes were analyzed by metascape website.Finally,RRM2 was selected for verification in hepatocellular carcinoma tissues and adjacent tissues,and it was found that the staining score of RRM2 in hepatocellular carcinoma tissues was(10.9±1.5)points,which was significantly higher than its staining score in adjacent tissues[(4.5±1.2)points],P<0.001.Conclusion The nine genes identified by bioinformatics analysis may be the key genes in the occurrence and development of hepatocellular carcinoma,which can provide reference for further study on the pathogenesis,diagnosis and treatment of hepatocellular carcinoma.