地皮菜抗三阴性乳腺癌作用活性成分的发现与靶点验证

Discovery and Target Verification of Active Ingredients of Nostoc Commune in Anti-triple-negative Breast Cancer

目的采用网络药理学方法和分子生物学实验探究地皮菜抗三阴性乳腺癌(triple-negative breast cancer,TNBC)的活性成分与靶点。方法查阅文献并结合实验室前期研究收集地皮菜的活性成分,采用Swiss Target Prediction数据库进行靶点预测,TTD、Genecards和OMIM数据库获取TNBC靶点。应用STRING在线平台进行蛋白互作,使用Metascape数据库进行KEGG信号通路和GO基因功能富集分析。通过AutoDock软件将地皮菜成分N-乙酰基色胺与靶点AKT1进行分子对接。采用Annexin V-FITC/PI双染色法检测地皮菜活性成分对乳腺癌细胞凋亡的影响。利用RT-qPCR和Western blotting检测地皮菜活性成分抗TNBC的作用机制。结果网络药理学结果发现N-乙酰基色胺、Scytonemin和Nostocionone等8个有效成分,涉及细胞信号传导和AKT1、STAT3、CCND1等75个关键靶点。KEGG信号通路和GO基因功能富集分析结果涉及癌症相关信号通路、PI3K-Akt信号通路和MAPK信号通路等。分子对接显示N-乙酰基色胺与AKT1的亲和作用较好。N-乙酰基色胺对乳腺癌细胞没有明显的促凋亡作用,Western blotting结果显示N-乙酰基色胺下调了AKTI的蛋白表达量。RT-qPCR结果显示N-乙酰基色胺可以有效降低乳腺癌细胞AKT1的mRNA表达。结论N-乙酰基色胺可能通过抑制AKT1信号通路抑制TNBC细胞增殖,从而发挥抗TNBC作用。

OBJECTIVE To explore the mechanism of action of active components of Nostoc commune in anti-triple-negative breast cancer(TNBC)by the network pharmacology method and molecular biology experiment.METHODS The active components of Nostoc commune were collected by consulting the literature and combined with the preliminary research in the laboratory,the Swiss Target Prediction database was used for target prediction,and the disease targets were obtained in the TTD,Genecards and OMIM databases.The STRING online platform was used for protein-protein interaction,and the KEGG signaling pathway and GO gene function enrichment analysis were performed using the Metascape database.Molecular docking of N-acetyltryptamine,a component of Nostoc commune,and target AKT1 by AutoDock software.Annexin V-FITC/PI double staining method was performed to analyze the apoptotic rate of cells.RT-qPCR and Western blotting were used to detect the mechanism of action of the active components of Nostoc commune on anti-TNBC.RESULTS The results of network pharmacology showed that there were 8 effective components,such as N-acetyltryptamine,Scytonemin and Nostocionone,involved 75 key targets such as signal transduction and AKT1,STAT3 and CCND1.The KEGG signaling pathway and GO gene function enrichment analysis results involved cancer-related signaling pathways,PI3K-Akt signaling pathways and MAPK signaling pathways.Molecular docking showed that N-acetyltryptamine had better affinity with AKT1.N-acetyltryptamine could not significantly promote apoptosis of breast cancer cells.Western blotting showed that N-acetyltryptamine could down-regulate the protein expressions of AKT1.The results of RT-qPCR showed that N-acetyltryptamine could effectively reduce the mRNA expression of AKT1 in cells.CONCLUSION N-acetyltryptamine may inhibit the proliferation of TNBC cells by inhibiting the AKT1 signaling pathway,thereby exerting anti-TNBC effects.