| Objective:The aim of this study is to investigate the mechanism of different concentrations of ursolic acid in hypopharyngeal carcinoma Fadu cells,and to verify the differences in the biological functions of the cells by cell proliferation,migration,and apoptosis.Secondly,according to gene sequencing,the potential mechanism is preliminarily explored,so as to provide new ideas for the drug treatment of hypopharyngeal carcinoma.Methods:Different concentrations of ursolic acid(0,5,10,15,20,30,40,50μmol/L)were applied to hypopharyngeal carcinoma Fadu cells.CCK-8 method was used to test the effects of ursolic acid on the activity of hypopharyngeal carcinoma Fadu cells.Clonal formation assay was used to detect the effects of ursolic acid(0,10,20μmol/L)on proliferation of Fadu cells.The effects of ursolic acid(0,10,20μmol/L)on cell migration were tested by cell scratch assay.The effect of flow cytometry on apoptosis;The expression of related pathway proteins was verified by Western-blot assay.RNA-seq technique was used to analyze the transcriptome of ursolic acid treated cells and screen differentially expressed genes.Results:With the increasing concentration of ursolic acid,CCK-8 assay showed that the absorbance value of Fadu cells decreased gradually,and the survival number of FadU cells decreased significantly.After 24 h,the concentration of IC50 was 22.45μmol/L.The results of cell clonal formation experiment showed that the number of cell clonal formation decreased with the increasing concentration of ursolic acid.The cell scratch test indicated that the migration ability of Fadu cells decreased with the increase of ursolic acid concentration.Flow cytometry showed that the apoptosis rate increased with the increase of ursolic acid concentration.Western blot results showed that ursolic acid could down-regulate the expression of MMP-9 and N-cadherin,up-regulate the expression of E-cadherin,and inhibit the epithelial mesenchymal transformation.Ursolic acid may inhibit cell proliferation and migration and induce cell apoptosis by inhibiting PI3K/Akt signaling pathway related proteins Akt and PI3K phosphorylation.RNA-Seq analysis showed that compared with control group,UA treatment group effectively down-regulated 113 genes in Fadu cells and up-regulated 179 genes in Fadu cells.Conclusion:1.Ursolic acid can inhibit the proliferation of hypopharyngeal carcinoma Fadu cells,and then inhibit the tumor progression.2.Ursolic acid decreased the migration ability of hypopharyngeal carcinoma Fadu cells.3.Ursolic acid inhibits EMT in hypopharyngeal carcinoma Fadu cells.4.Ursolic acid induced apoptosis of hypopharyngeal carcinoma Fadu cells.5.Ursolic acid inhibited the phosphorylation of PI3K and Akt in hypopharyngeal carcinoma Fadu cells.6.Ursolic acid changes the expression of related genes in hypopharyngeal carcinoma Fadu cells. |
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