基于网络药理学与体外实验探讨淫羊藿素抗非小细胞肺癌EGFR-TKIs耐药的分子机制

Exploring molecular mechanism of icaritin against non⁃small cell lung cancer EGFR⁃TKIs resistance based on network pharmacology and in vitro experiments

目的基于网络药理学与体外实验探讨淫羊藿素抗非小细胞肺癌(NSCLC)表皮生长因子受体-酪氨酸酶抑制剂(EGFR-TKIs)耐药的分子机制。方法利用PubChem和化合物靶点预测(Swiss Target Prediction)数据库下载淫羊藿素的简化分子线性输入规范(SMILES)号及作用靶点,通过人类基因(GeneCards)和在线人类孟德尔遗传(OMIM)数据库收集NSCLC耐药疾病靶点,将药物与疾病交集靶点导入STRING数据库分析蛋白质-蛋白质相互作用(PPI)情况,运用Cytoscape 3.9.1软件内置插件计算节点拓扑参数值并筛选核心靶点,采用生物信息学分析平台(DAVID)数据库进行京都基因与基因组百科全书(KEGG)和基因本体(GO)富集分析,构建“淫羊藿素-关键靶点-疾病-通路”图。使用Pymol和Autodock Tools 1.5.7软件进行分子对接。体外实验选用NSCLC耐药株PC9OR研究淫羊藿素对细胞增殖、集落形成、迁移、侵袭和凋亡能力的影响,并对富集得到的核心靶点及关键通路进行验证。结果共筛选到淫羊藿素治疗NSCLC耐药的潜在作用靶点1952个。通过PPI网络节点拓扑参数值筛选得到13个核心靶点,涉及蛋白激酶B(AKT1)、雌激素受体α(ESR1)、B淋巴细胞瘤2(BCL2)、表皮生长因子受体(EGFR)等基因。KEGG通路富集分析显示,癌症相关通路、磷脂酰肌醇-3-激酶-蛋白激酶B(PI3K-AKT)信号通路、EGFR-TKIs耐药通路等可能在淫羊藿素治疗NSCLC EGFR-TKIs耐药的过程中起关键作用。GO富集分析显示,细胞功能涉及信号传导、凋亡过程的负调控、DNA转录的正调控等。分子对接显示淫羊藿素与各核心靶点均具有较强的结合能力。细胞实验表明,淫羊藿素抑制耐药细胞的增殖、集落形成、迁移、侵袭及促进细胞凋亡,并下调ESR1、AKT1、EGFR等mRNA表达水平以及PI3K-AKT通路磷酸化磷脂酰肌醇-3-激酶(p-PI3K)、磷酸化蛋白激酶B(p-AKT)的关键蛋白水平。结论淫羊藿素可能通过多靶点调控PI3K-AKT通路抑制EGFR-TKIs耐药细胞的增殖、集落形成、迁移、侵袭及促进细胞凋亡,从而发挥抗NSCLC EGFR-TKIs耐药的作用。

Objective To explore the molecular mechanism of icaritin against the resistance to epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)in non-small cell lung cancer(NSCLC)based on network pharmacology and in vitro experiments.Methods Download the Simplified Molecular Input Line Entry System(SMILES)notation and therapeutic targets of icaritin from PubChem and Swiss Target Prediction databases.Collect NSCLC resistance disease targets using the GeneCards and Online Mendelian Inheritance in Man(OMIM)databases.Import the intersecting targets of the drug and disease into the STRING database to analyze protein-protein interactions(PPI).Use the Cytoscape 3.9.1 software with built-in plugins to calculate node topological parameters and filter core targets.Perform Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)enrichment analysis using the Database for Annotation,Visualization,and Integrated Discovery(DAVID),and construct the"icaritin-key target-disease-pathway"map.Conduct molecular docking using Pymol and Autodock Tools 1.5.7 software.In the in vitro experiments,NSCLC resistant cell line PC9OR was used to study the effects of icaritin on cell proliferation,colony formation,migration,invasion,and apoptosis,and to verify the core targets and key pathways obtained from the enrichment analysis.Results A total of 1952 potential targets for icaritin in treating NSCLC resistance were identified.Through the analysis of topological parameters in the PPI network,13 core targets were screened,including genes such as protein kinase B(AKT1),estrogen receptorα(ESR1),B-cell lymphoma 2(BCL2),and epidermal growth factor receptor(EGFR).KEGG pathway enrichment analysis indicated that cancer-related pathways,the phosphatidylinositol-3-kinase(PI3K)-protein kinose 13(AKT)signaling pathway,and the EGFR-TKIs resistance pathway might play crucial roles in the treatment of NSCLC EGFR-TKIs resistance by icaritin.GO enrichment analysis showed that cellular functions involved signal transduction,negative regulation of apoptosis,and positive regulation of DNA transcription.Molecular docking revealed that icaritin had strong binding affinities with each core target.Cell experiments demonstrated that icaritin inhibited the proliferation,colony formation,migration,invasion of resistant cells,and promoted apoptosis of resistant cells,while downregulating the mRNA expression levels of ESR1,AKT1,and EGFR,as well as the key protein levels of phosphorylated PI3K(p-PI3K)and phosphorylated AKT(p-AKT)in the PI3K-AKT pathway.Conclusions Icaritin may exert its anti-NSCLC EGFR-TKIs resistance effect by multi-target regulation of the PI3K-AKT pathway,thereby inhibiting the proliferation,colony formation,migration,invasion of resistant cells,and promoting the apoptosis of EGFR-TKIs resistant cells.