基于加权基因共表达网络分析识别F2RL1为结直肠癌的核心致病基因

Identification of F2RL1 as a Causative Hub Gene of Colorectal Cancer Based on Weighted Gene Co-expression Network Analysis

目的通过生物信息学方法鉴定与结直肠癌相关的核心基因,并初步验证。方法从TCGA数据库中下载正常人和结肠腺癌(COAD)患者的临床资料和基因表达数据;从GEO数据库中下载275例结直肠癌患者的肿瘤组织及其癌旁正常组织基因表达数据。通过差异表达基因分析和加权基因共表达网络分析(WGCNA)筛选差异共表达基因。对差异共表达基因进行GO和KEGG分析,并通过STRING数据库构建蛋白质互作(PPI)网络,最后利用Cytoscape插件CytoHubba提取候选核心基因。将与总生存期(OS)相关的核心基因通过人类蛋白图谱(HPA)数据库验证其蛋白表达,并通过单基因GSEA探索核心基因相关的信号通路。通过Western blot法在人正常结肠上皮细胞HCoEpiC和结直肠癌细胞HCT116、LoVo中验证生信分析的结果。结果通过对TCGA和GSE89076数据集的WGCNA和差异表达基因分析识别到436个差异共表达基因,用R语言clusterProfiler包进行功能和通路富集分析:GO分析结果显示,这些基因在714个生物过程、45个细胞组成和153个分子功能中富集;KEGG分析结果显示,这些基因在9条通路中富集。在436个差异共表达基因的PPI网络中鉴定了10个候选核心基因(BDKRB2、GCG、EDN2、EDN3、P2RY2、P2RY1、F2RL1、GNA11、SST、ADRA2)。通过GEPIA2在线工具对候选核心基因的预后价值进行验证,发现F2RL1与COAD患者OS相关。经HPA数据库验证,F2RL1蛋白表达水平在结直肠癌样本中下调,这与其mRNA水平的改变一致。单基因GSEA分析提示F2RL1与结直肠癌的发生发展密切相关。Western blot实验证明F2RL1在人结直肠癌细胞系中表达下调。结论F2RL1可作为结直肠癌的候选生物标志物,本研究为结直肠癌的诊断、预后及靶向治疗提供了新线索。

Objective To identify hub genes related to colorectal cancer by using bioinformatics methods and verify them primarily.Methods Clinical data and gene expression data of normal colon and colon adenocarcinoma(COAD)were downloaded from the TCGA database.Meanwhile,paired normal and tumor tissues from 275 patients with colorectal cancer were obtained from the GEO database.The differential co-expressed genes were screened out by differential gene expression analysis and weighted gene co-expression network analysis(WGCNA).These genes were analyzed by GO and KEGG,and the protein-protein interaction(PPI)network was constructed using String database.Finally,the hub genes were identified by using the CytoHubba plugin of Cytoscape.The protein expression of hub gene related to overall survival(OS)was verified by the Human Protein Atlas(HPA)database,and the signaling pathways related to hub gene were explored by using single gene GSEA.The results from bioinformatics analysis were verified by Western blotting in human normal colonic epithelial cells HCoEpiC,colorectal cancer cells HCT116,and LoVo.Results There were 436 genes identified differentially through WGCNA and differential analysis from TCGA and GSE89076.The functional and pathway enrichment analysis were carried out by using R clusterProfiler package.The results of GO analysis showed that these genes were enriched in 714 biological processes,45 cell compositions,and 153 molecular functions;KEGG analysis showed that these genes were enriched in 9 pathways.Then we identified 10 candidate hub genes in the PPI network of 436 genes(BDKRB2,GCG,EDN2,EDN3,P2 RY2,P2 RY1,F2 RL1 GNA11,SST,ADRA2).The prognostic value of the candidate hub genes was verified by GEPIA2 online tool and F2 RL1 was found to be associated with OS.F2 RL1 protein levels verified in the HPA database were downregulated in colorectal cancer samples,which were consistent with the changing of mRNA levels.Single-gene GSEA analysis suggested that F2 RL1 was associated with development of colorectal cancer.Western blotting verified that F2 RL1 expression levels were down-regulated in human colorectal cancer cell lines.Conclusion F2 RL1 was identified as a candidate biomarker for colorectal cancer,which may provide theoretical basis for the diagnosis,prognosis,and targeted therapy of colorectal cancer.