Exploring The Effect And Mechanism Of Astragaloside Ⅳ On Hepatocellular Carcinoma Based On Bioinformatics Analysis And Experimental Study

OBJECTIVE: Based on bioinformatics study,we analyzed the core targets and key signaling pathways of Astragaloside IV in hepatocellular carcinoma and investigated the effects of Astragaloside IV on proliferation,migration,invasion,cycle,apoptosis and core gene and protein expression of Hep G2 and Huh7 cells in combination with in vitro experiments.METHODS:1.The 2D structure of Astragaloside IV(AS-IV)was obtained using Pub Chem Bio Assay database,and the 2D structure was converted into 3D structure using Chem Bio3 D MLtra 14.0;Search for drug targets in Gene Cards,Swisstarget prediction,pharmmapper database and disease targets in TCGA database to make Venn diagrams.Combined with String database and Cytoscape 3.7.0 software to build "drug-target-disease" network pharmacology map.Using DAVID database to do GO and KEGG enrichment analysis of common targets and make it is to be visualized.Protein-protein interaction(PPI)network was used to analyze the core targets of Astragaloside IV to hepatocellular carcinoma.Establishing a "drug-protein" molecular docking model between Astragaloside IV and the core target of hepatocellular carcinoma using molecular docking technology.Molecular dynamics simulation was used to construct a molecular dynamics model of Astragaloside IV and core targets to verify the feasibility of molecular docking;Combined with PEGIA database and CIBERSORT R script v1.03 software to analyze the co-expression of core genes with immune cells and immune infiltration.2.Both conventionally cultured Hep G2 and Huh7 cells were divided into control group(DMEM group)and AS-IV group(DMEM+AS-IV groups,divided into low,medium and high concentration groups).Concentrations of 0μg/m L,40μg/m L,80μg/m L,120μg/m L,160μg/m L,180μg/m L Astragaloside IV were taken to act on Hep G2 and Huh7 cells respectively.The effect of AS-IV on the value-added of Hep G2 and Huh7 cells was detected by CCK8 after 24 h,48h and 72 h,respectively.Wound healing assay,clone formation assay and invasion assay to analyze the effects of AS-IV on migration,proliferation and invasion of Hep G2 and Huh7 cells.The effect of AS-IV on the cell cycle and apoptosis of Hep G2 and Huh7 cells was detected by flow cytometry;the relative expression of core genes was detected by q RT-PCR.Western blot was used to detect the expression of core proteins.RESULTS:1.PPI results showed that: AS-IV acted on the core gene of hepatocellular carcinoma as VEGFA.Molecular dynamics simulation of AS-IV and VEGFA protein verified the feasibility of molecular docking.The highest correlation of VEGFA gene expression in hepatocellular carcinoma tissues,normal liver tissues,and hepatocellular carcinoma parietal tissues were M0 macrophages,M2 macrophages,regulatory T cells,monocytes,and mast cells,and VEGFA gene expression in hepatocellular carcinoma tissues was also significantly different compared with lymphatic tissues,blood vessels,ascites and normal liver tissues;the expression of naive CD4 T cells,resting memory CD4 T cells,activated NK cells,activated dendritic cells,and activated mast cells was the most significant in immune infiltration of hepatocellular carcinoma(P<0.05).The VEGFA gene in hepatocellular carcinoma tumor mutational load was negatively correlated with its methylation status was negatively correlated(P>0.05)and TGF-β1 was positively correlated with its methylation status(P<0.05);KEGG enrichment analysis showed that the enrichment pathways of AS-IV in hepatocellular carcinoma were VEGF signaling pathway(P<0.05)and TGF-β1-based hepatocellular carcinoma signaling pathway(hsa05225)(P<0.05).2.In vitro experimental studies showed that when the concentration of AS-IV was 180 μg/m L and the intervention time was 48 h,the wound healing assay and invasion assay suggested that AS-IV inhibited the migration and invasion ability of Hep G2 and Huh7 cells most significantly(P < 0.05,P < 0.05);when the concentration of AS-IV was 180 μg/m L and the intervention time was 14 days,the clone formation assay suggested that AS-IV inhibited the proliferation of Hep G2 and Huh7 cells most significantly(P < 0.05).The flow cytometry results showed that AS-IV could inhibit Hep G2 and Huh7 cells from G1 to G2 phase(P<0.05)and could promote their apoptosis(P<0.05);q RT-PCR results showed that AS-IV could down-regulate VEGFA expression and up-regulate TGF-β1 expression(P<0.05,P<0.05);Western blot results showed that AS-IV had the most significant effect on Hep G2 and Huh7 cells VEGFA protein expression was inhibited and TGF-β1 protein expression was promoted(P < 0.05,P < 0.05).CONCLUSION: Astragaloside IV has the ability to inhibit the migration,invasion and proliferation of hepatocellular carcinoma cells and promote their apoptosis,and the mechanism may be related to the regulation of VEGFA and TGF-β1expression through multi-target and multi-pathway.