To Explore The Mechanism Of Kaempferol And Formononetin Against Non-small Cell Lung Cancer Based On Network Pharmacology

Objective Based on network pharmacology and bioinformatics,this study explored the anti-non-small cell lung cancer effects and mechanisms of Kaempferol and formononetin by in vitro cell experiments.Method(1)To explore the molecular mechanism of kaempferol and formononetin on non-small cell lung cancer through network pharmacology.Firstly,the targets of kaempferol and formononetin were obtained from Swiss Target Prediction and Pharmapper databases,the targets of non-small cell lung cancer were obtained from Gene Cards database and TTD database,the differential genes of non-small cell lung cancer were obtained from GEO database by using information generation technology,and the intersection targets of kaempferol,formononetin and non-small cell lung cancer were obtained from Wayne platform.Using Cytoscape3.9.0 software to draw PPI network and obtain the core target;GO and KEGG enrichment analysis were carried out by using DAVID database,and the "target-pathway" map was drawn by Cytoscape3.9.0 software.Using GEPIA,HPA and Kaplan-Meier Plotter databases,the gene expression level analysis,protein expression level analysis and survival analysis of core targets were carried out respectively.(2)To investigate the effects of Kaempferol and formononetin on proliferation,migration,invasion and apoptosis of lung cancer A549 cells in vitro.The effects of kaempferol and formononetin on the proliferation of lung cancer A549 cells were investigated by CCK-8 and plate clone formation experiments,and the half inhibition rate(IC50)was calculated according to the experimental results of CCK-8 to determine the dosage.The effects of kaempferol and formononetin on the adhesion of lung cancer A549 cells were detected by cell adhesion test.The effects of kaempferol and formononetin on the migration of lung cancer A549 cells were detected by scratch test.Transwell experiment was used to detect the effects of kaempferol and formononetin on the invasion of lung cancer A549 cells.Hoechst33342 staining was used to detect the effects of kaempferol and formononetin on the apoptosis of lung cancer A549 cells.q PCR experiment was used to detect the effects of kaempferol and formononetin on the m RNA expression levels of PI3 K,Akt,Bcl-2 and Bax in lung cancer A549 cells.The effects of kaempferol and formononetin on the expression levels of PI3 K,Akt,p-Akt,Bcl-2 and Bax proteins in lung cancer A549 cells were detected by Western Blot.Results(1)A total of 113 targets of kaempferol and formononetin were screened out by Swiss Target Prediction and Pharmapper databases,5,818 targets of non-small cell lung cancer were obtained by Gene Cards and TTD databases,and 3,812 differential genes were obtained from GEO database by using information generation technology,including 1,652up-regulated genes,2,160 down-regulated genes and 83 cross targets by Wayne platform.The top five core targets were AKT1,ESR1,SRC,EGFR and MMP9 screened by Cyto Hubba,the plug-in of Cytoscape3.9.0 software,and the top one was AKT1.The top five in KEGG enrichment analysis are endocrine resistance,nitrogen metabolism,EGFR tyrosine kinase inhibitor resistance,cancer pathway and steroid hormone biosynthesis,all of which are closely related to PI3K/Akt signaling pathway.In the ’ target-pathway ’ network diagram,the genes most associated with the pathway were PIK3R1 and AKT1,and the signal pathway most associated with the gene was PI3 K / Akt.These results indicate that the main signaling pathway of kaempferol and formononetin in non-small cell lung cancer may be PI3 K / Akt.(2)The effects of Kaempferol and formononetin on lung cancer A549 cells were studied in cell experiments.The results of CCK-8 and plate clone formation experiments showed that Kaempferol and formononetin could significantly inhibit the proliferation of lung cancer A549 cells in a concentration-dependent and time-dependent manner.The results of cell adhesion experiment showed that kaempferol and formononetin could significantly inhibit the adhesion of lung cancer A549 cells.The results of scratch test showed that Kaempferol and formononetin could significantly inhibit the migration of lung cancer A549 cells.Transwell experiment showed that kaempferol and formononetin could significantly inhibit the invasion of lung cancer A549 cells.Hoechst33342 staining showed that kaempferol and formononetin could promote the apoptosis of lung cancer A549 cells.QPCR results showed that kaempferol and formononetin could down-regulate the m RNA expression levels of PI3 K,Akt and Bcl-2 in A549 cells of lung cancer,and up-regulate the m RNA expression level of Bax.Western Blot results showed that kaempferol and formononetin could down-regulate the protein expression levels of PI3 K,Akt,p-Akt and Bcl-2 in A549 cells of lung cancer,and up-regulate the protein expression level of Bax.Conclusion Kaempferol and formononetin can significantly inhibit the proliferation,migration and invasion of lung cancer A549 cells,and promote their apoptosis,which may be through inhibiting the activation of PI3K/Akt signaling pathway.