基于WGCNA联合网络药理学探讨益髓生血胶囊治疗肝细胞癌的分子机制

Molecular Mechanism of Benefiting-marrow Generating-blood Capsules in the Treatment of Hepatocellular Carcinoma Based on WGCNA and Network Pharmacology

目的:基于加权基因共表达网络分析(weighted gene co-expression network analysis,WGCNA)联合网络药理学方法探讨益髓生血胶囊治疗肝细胞癌(hepatocellular carcinoma,HCC)的分子机制,筛选肝细胞癌潜在预后标志物。方法:采用中药系统药理学数据库与分析平台(traditional Chinese medicine systems pharmacology database and analysis platform,TCMSP)筛选益髓生血胶囊有效成分对应靶点;从TCGA数据库下载HCC及癌旁组织基因芯片,通过“Limma”包筛选差异基因;使用WGCNA包通过TCGA数据库中HCC的基因表达数据图谱构建基因共表达网络,筛选与临床性状相关基因模块;对益髓生血胶囊调控基因、HCC差异基因、模块基因取交集;使用Cytoscape构建药物-靶点-疾病网络,对交集基因进行基因本体论(gene ontology,Go)和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路富集分析;使用STRING数据库构建交集基因间的蛋白-蛋白互作(protein-protein interactions,PPI)网络;根据最大团中心性(maximal clique centrality,MCC)评分对前10个基因与HCC的预后进行探索,进一步分析以获得HCC潜在标志物。结果:共获得益髓生血胶囊438个有效成分对应靶点;从TCGA数据库共获得374个肿瘤样本和50个正常样本,Limma包共筛选出2703个差异基因;WGCNA筛选出1922个与正常组织相关的基因,交集基因共26个,主要富集于细胞衰老、细胞周期、乙肝、p53信号通路、多种癌症、IL-17等信号通路;26个交集基因中,7个基因的高表达与肝癌患者的较差总生存率(overall survival,OS)/无病生存率(disease free survival,DFS)具有显著差异(P<0.05)。结论:益髓生血胶囊治疗HCC的作用机制复杂,主要涉及细胞衰老、细胞周期、乙肝、p53信号通路、多种癌症、IL-17等信号通路,其主要作用成分可能与榭皮素、木草素、芦荟大黄素、芒柄花素等有关。BIRC5、CCNA2、CCNB1、CDK1、CHEK1、E2F1、TOP2A等7个基因可能为HCC的潜在预后生物标志物。

Objective:To discuss molecular mechanism of Yisui Shengxue(Benefiting-marrow Generatingblood)capsules in the treatment of hepatocellular carcinoma on the foundation of weighted gene co-expression network analysis(WGCNA)and network pharmacology,and to screen for potential prognostic markers in hepatocellular carcinoma.Methods:TCMSP was adopted to screen the corresponding targets of the active ingredients of the capsules;the gene chips of hepatocellular carcinoma and paraneoplastic tissue were downloaded from TCGA database,and the differential genes were screened from Limma package;the WGCNA package was used to construct the gene co-expression network for the gene expression data map of hepatocellular carcinoma in TCGA database,and screen the gene modules related to clinical traits;the intersection of benefiting-marrow generatingblood capsules regulatory genes,hepatocellular carcinoma differential genes and module genes were taken to construct the internet of drug-target-disease by Cytoscape,GO and KEGG pathway enrichment analysis of the intersected genes were conducted;STRING database was adopted to construct protein-protein interactions(PPI)network;the top ten genes and the prognosis of hepatocellular carcinoma were surveyed according to maximal clique centrality(MCC)scales,in order to obtain the potential markers of hepatocellular carcinoma by further analysis.Results:The analysis has yielded 438 corresponding targets of active ingredients of the capsules;374 tumor samples and 50 normal samples were obtained from TCGA database,and 2703 differential genes were gained from Limma package;1922 genes related to normal tissues and 26 intersected genes were screened by WGCNA,mainly concentrated in cell senescence,cell cycle,hepatitis B,p53 signaling pathway,many kinds of cancer,IL-17 and other signling pathways;among the 26 intersection genes,the significant differences existed between the high expression of seven genes and the poor overall survival(OS)/disease free survival(DFS)in patients with liver cancer(P<0.05).Conclusion:The complex mechanism of the capsules in the treatment of hepatocellular carcinoma mainly involves cellular aging,cell cycle,hepatitis B,p53 signaling pathway,many kinds of cancer,IL-17 and other signling pathways,and its main active ingredients are likely to related to quercetin,luteolin,aloe emodin and formononetin.Seven genes including BIRC5,CCNA2,CCNB1,CDK1,CHEK1,E2F1 and TOP2A may be the potential prognostic biomarkers of hepatocellular carcinoma.