基于加权基因共表达网络分析探究养肺方治疗非小细胞肺癌的作用机制及实验验证

Action Mechanism of Lung Nourishment Formula inTreatment of Non-small Cell Lung Cancer Based on Weighted Gene Co-expression Network

目的:基于网络药理学联合加权基因共表达网络分析(weighted correlation network analysis,WGCNA)及实验验证初步探究养肺方治疗非小细胞肺癌(non-small cell lung cancer,NSCLC)的作用机制。方法:在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)筛选养肺方组方中药的化学成分。利用中医药百科全书(The encyclopedia of traditional Chinese medicine,ETCM)预测养肺方活性成分相关靶点。运用GEO平台下载基因芯片GSE118370,采用GEO2R归一化数据并分析差异基因。采用WGCNA进行基因模块分析并筛选疾病靶点基因。利用Cytoscape 3.7.2软件构建的“药物-活性成分-靶点”网络并进行拓扑分析筛选有效药效成分。将WGCNA筛选出核心基因与药物靶点取交集得到治疗靶点,并构建蛋白质互作(protein-protein interaction,PPI)网络,通过Cytoscape 3.7.2中的CytoHubba筛选出核心治疗靶点。利用R软件进行基因本体(gene ontology,GO)功能富集分析和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)信号通路富集分析。40只雄性Wistar大鼠随机分为养肺方组(400 g·kg-1)和空白组,每组20只,大鼠连续灌胃给药7天,每天2次,灌胃结束后腹主动脉取血制备含药血清。采用不同浓度含药血清干预人肺腺癌PC9细胞株后,CCK-8试剂检测细胞活力;Western Blot检测p-蛋白激酶B(protein kinase B,AKT)、p-磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinases,PI3K)蛋白表达水平;实时定量荧光PCR分析PI3K mRNA、AKT mRNA表达水平;TUNEL检测细胞凋亡水平;结果:在TCMSP数据库中筛选出65个养肺方活性成分;通过ETCM数据库获得283个药物靶点。利用GEO2R分析得到差异基因1192个。WGCNA基因模块分析得到核心基因30个,与药物靶点取交集,获得交集靶点基因10个。PPI网络分析得到核心靶点5个。富集分析得到939个GO条目及82条信号通路。CCK-8检测发现养肺方含药血清浓度为10%时,细胞活力最差。与空白组比较,养肺方组p-PI3K、p-AKT蛋白表达水平及PI3K、AKT的mRNA表达水平明显降低(P<0.05),TUNEL染色阳性细胞数显著增加(P<0.05)。结论:养肺方可能通过抑制PI3K/AKT通路诱导NSCLC肿瘤细胞凋亡,进而改善NSCLC。

Objective:To investigate the mechanism of non-small cell lung cancer(NSCLC)based on weighted correlation network analysis(WGCNA)and experimental verification.Methods:The chemical components of traditional medicine formulas were screened in the traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP).The encyclopedia of traditional Chinese medicine(ETCM)was used to predict the targets related to the active ingredients of lung nourishment.The GEO platform was used to download the gene chip GSE118370,and the GEO2R normalized data was used to analyze the differential genes.WGCNA was used for gene module analysis and screening of disease target genes.The"drug-active ingredient-target"network built by Cytoscape 3.7.2 software was used and topological analysis was performed to screen for effective pharmacodynamic ingredients.WGCNA screened out the core gene and drug target to obtain the therapeutic target,and the protein-protein interaction(PPI)network was constructed,and the core therapeutic target was screened out by CytoHubba in Cytoscape 3.7.2.R software was used to perform gene ontology(GO)functional enrichment analysis and kyoto encyclopedia of genes and genomes(KEGG)signaling pathway enrichment analysis.Forty male Wistar rats were randomly divided into lung feeding group(400 g·kg-1)and blank group,20 rats in each group,rats were continuously administered by gavage for 7 days,twice a day,and after the end of gavage,the abdominal aorta blood was taken to prepare drug-containing serum.After different concentrations of drug-containing serum were used to intervene in human lung adenocarcinoma PC9 cell line,CCK-8 reagent detected cell viability.Western Blot measured the expression levels of p-protein kinase B(AKT)and p-phosphatidylinositol 3-kinases(PI3K).Real-time quantitative fluorescence PCR analysis of PI3K mRNA and AKT mRNA expression levels;TUNEL detects apoptosis levels;Results:65 active ingredients of lung nourishment formula were screened out in TCMSP database.283 drug targets were obtained through the ETCM database.GEO2R analysis was used to obtain 1192 differential genes.The WGCNA gene module analysis obtained 30 core genes,which intersected with drug targets to obtain 10 intersection target genes.PPI network analysis yielded 5 core targets.The enrichment analysis yielded 939 GO entries and 82 signal pathways.CCK-8 detection found that cell viability was the worst when the serum concentration of the drug contained in the lung nourishing formula was 10%.Compared with the control group,the expression levels of p-PI3K and p-AKT protein and the mRNA expression levels of PI3K and AKT were significantly reduced in the lung nourishing group(P<0.05),the number of cells positive for TUNEL staining increased significantly(P<0.05).Conclusion:Lung nourishment may induce apoptosis of NSCLC tumor cells by inhibiting the PI3K/AKT pathway,thereby improving NSCLC.