This study aimed to explore the effects and mechanisms of isorhamnetin (IH) on nasopharyngeal carcinoma (NPC) cells.The NPC cell line C666-1 was treated with different concentrations of IH to investigate its effects on cell proliferation,migration,invasion,apoptosis,and cell cycle.Network pharmacology and molecular docking approaches were adopted to predict the action targets and signaling pathways of IH.The disease-related targets of NPC were obtained from the DisGeNET,GeneCards,and OMIM databases,and the potential action targets of IH were obtained from the SwissTargetPrediction database.To identify the core targets and pathways of IH in acting on NPC,multiple bioinformatics tools and databases,including Venny,Cytoscape 3.10.1,String,and Metascape,were employed.Molecular docking was further conducted to validate the binding affinity between IH and the core targets.Subsequently,transcriptome sequencing was performed to analyze the differentially expressed genes and perturbed signaling pathways in C666-1 cells following IH treatment.Finally,the protein expression levels of the action targets and key components of the signaling pathways were detected by Western blot.The results showed that IH could inhibit the proliferation,migration,and invasion abilities of C666-1 cells in a concentration-dependent manner.Also IH significantly promoted apoptosis and arrested the cell cycle at the G1 phase.Network pharmacology analysis predicted that clusterin (CLU),protein kinase B (AKT),epidermal growth factor receptor (EGFR),and matrix metalloproteinase 9 (MMP9) were the core action targets of IH.Molecular docking results indicated that IH exhibited the most stable binding to CLU( binding energy:-9.1 kcal/mol ).KEGG pathway enrichment analysis revealed that the signaling pathways significantly affected by IH mainly included the phosphoinositide 3-kinase (PI3K)/AKT pathway,EGFR tyrosine kinase inhibitor resistance pathway,and several cancer-related pathways.Results of transcriptome sequencing demonstrated that the PI3K/AKT pathway exhibited the most significant changes among the top 20 differentially-regulated pathways.Western blot analysis confirmed that the protein expression levels of CLU,phosphorylated PI3K (p-PI3K),and phosphorylated AKT (p-AKT) decreased in a dose-dependent manner after IH treatment.In conclusion,this study provides evidence that IH exerts inhibitory effects on NPC cells by suppressing the CLU and PI3K/AKT signaling pathways.