摘要
目的基于网络药理学研究香菇多糖抑制三阴乳腺癌(TNBC)的作用机制并采用细胞和动物实验进行验证。方法通过GeneCards数据库和DisGeNET数据库筛选与TNBC相关基因靶点,利用TCMID、PubChem、SwissTargetPrediction和GeneCards数据库查询香菇多糖相关基因靶点。使用Sangerbox软件进行基因本体论(GO)富集和京都基因与基因组百科全书(KEGG)通路富集分析。结合STRING数据库与Cytoscape 3.7.0软件将共同靶点进行可视化处理,筛选核心靶点,构建"化合物-靶点-通路"网络。利用Metascape软件进行转录因子及相关调控基因特异富集。体外培养小鼠TNBC细胞4T1和人TNBC细胞MDA-MB-231,磺酰罗丹明B染色法观察香菇多糖(31.25、62.5、125、250、500、1000μg·mL^(-1))对细胞存活率的影响;健康雌性BABLC小鼠sc接种1×10^(6)个4T1-Luc细胞构建TNBC模型,通过小动物活体成像系统观察香菇多糖(100、200 mg·kg^(-1))对肿瘤生长的影响,实时荧光定量PCR(qRT-PCR)技术检测肿瘤组织信号转导和转录活化因子3(STAT3)和血管内皮生长因子A(VEGFA)mRNA表达。结果数据库及软件分析得到香菇多糖治疗TNBC关键靶点52个,靶点主要涉及PI3K-Akt、AGE-RAGE、HIF-1、MAPK信号通路和肿瘤蛋白多糖相关通路,PPI分析得到VEGFA、STAT3、MAPK1、IL2、TNF、RELA、AKT1、MAPK3、BCL2L1和HSP90AA110个hub基因。与对照组比较,香菇多糖对4T1和MDA-MB-231细胞存活率均有显著抑制作用(P<0.05、0.01),且作用呈浓度相关性;在给药14、21d后,与模型组比较,香菇多糖能够剂量相关性地抑制小鼠TNBC肿瘤的生长,高剂量组差异显著(P<0.05、0.01),21 d抑制率达到(91.9±4.7)%;与对照组比较,香菇多糖给药后能够剂量相关性抑制STAT3和VEGFA的mRNA表达,高剂量组差异显著(P<0.05、0.01)。结论香菇多糖可通过多靶点、多途径协同作用抑制TNBC的生长。
Objective To study the mechanism of lentinan on inhibiting triple negative breast cancer(TNBC)based on network pharmacology and to verify it by animal and molecular biology experiments.Methods TNBC related genes were identified used GeneCards database and DisGeNET database.Lentinan-related gene targets were inquired used TCMID,PubChem,SwissTargetPrediction and GeneCards database.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)signal pathway was analysed by Sangerbox software.Core targets were visualized by combining STRING database and Cytoscape 3.7.0 software,and"chemicals-targets-pathways"network was established.The transcription factors and related regulatory genes were specifically enriched by Metascape software.Mouse TNBC cells 4T1 and human TNBC cells MDA-MB-231 were cultured in vitro.The effects of lentinan(31.25,62.5,125,250,500,1000μg·mL−1)on cell viability were observed by sulfoylrhodamine B staining.The TNBC model was constructed by inoculated with 1×10^(6)4T1-Luc cells by sc of healthy female BABLC mice.The effect of lentinan(100 and 200 mg·kg^(−1))on tumor growth was observed by in vivo small animal imaging system.The mRNA expressions of signal transducer and activator of transcription 3(STAT3)and vascular endothelial growth factor A(VEGFA)in tumor tissues were detected by real-time fluorescent quantitative PCR(qRT-PCR).Results Totally 52 key targets of lentinan related to TNBC were identified by database and software analysis,which mainly involving PI3K-Akt,AGE-RAGE,HIF-1,MAPK and proteoglycans related pathways.Totally 10 hub genes including VEGFA,STAT3,MAPK1,IL2,TNF,RELA,AKT1,MAPK3,BCL2L1 and HSP90AA1 were identified by PPI analysis.Compared with the control group,lentinan significantly inhibited the survival rate of 4T1 and MDA-MB-231 cells(P<0.05,0.01),and the effect was concentration dependent.After 14 and 21 days of administration,lendinan could inhibit the growth of TNBC tumor in mice dose-dependent compared with the model group,and the difference was significant in the high-dose group(P<0.05,0.01),and the 21-day inhibition rate reached(91.9±4.7)%.Compared with the control group,lentinan administration could inhibit the mRNA expression of STAT3 and VEGFA in a dose-dependent manner,and the differences were significant in the high-dose group(P<0.05,0.01).Conclusion Lentinan can inhibit the growth of TNBC through the synergistic effect of multiple targets and multiple pathways.
作者
代晓阳
张燕
王红磊
赵家熙
杨晓春
翁勤洁
DAI Xiaoyang;ZHANG Yan;WANG Honglei;ZHAO Jiaxi;YANG Xiaochun;WENG Qinjie(Center for Drug Safety Evaluation and Research,College of Pharmaceutical Sciences,Zhejiang University,Hangzhou 310058,China)
出处
《药物评价研究》
CAS
2022年第10期2031-2038,共8页
Drug Evaluation Research
基金
浙江大学实验技术研究项目(SJS202016)。
关键词
香菇多糖
三阴乳腺癌
网络药理学
信号转导和转录活化因子3
血管内皮生长因子A
lentinan
triple negative breast cancer
network pharmacology
signal transducer and activator of transcription 3
vascular endothelial growth factor A
