Inhibitory Effect Of Genistein On Nasopharyngeal Carcinoma CNE1 Cells And Its Mechanism

Objective:In vitro and in vivo experiments were used to explore the inhibitory effect and mechanism of genistein（Gen）on human nasopharyngeal carcinoma CNE1 cells,providing a theoretical basis for the application of genistein in the treatment of nasopharyngeal carcinoma,and perfecting the inhibitory mechanism on nasopharyngeal carcinoma of Sophora tonkinensis Gagnep.Methods:1.The CKK-8 method,colony formation,migration,invasion,cell cycle and apoptosis experiments were used to detect the proliferation,colony formation,migration,invasion,cell cycle and cell proliferation,colony formation,migration,invasion,cell cycle and apoptosis effects of genistein on CNE1 cells.2.Through experiments of subcutaneous transplanted tumors in nude mice,the effects of different doses of genistein on the growth of nasopharyngeal carcinoma tumors in vivo were investigated.3.Intervene CNE1 cells with 30μmol/L genistein,find out the differential genes through high-throughput sequencing and bioinformatics analysis,and then use Real-time fluorescence quantitative polymerase chain reaction（RT-qPCR）and Western blot to verify the molecular targets of genistein against nasopharyngeal carcinoma.Results:1.The 50%inhibitory concentrations of genistein on nasopharyngeal carcinoma CNE1 cells at 24 h,48 h and 72 h determined by CKK-8 method were 56.32μmol/L,34.13μmol/L and 21.90μmol/L.Through cell clone formation experiments,the clone formation rate of each group was（67.33±6.40）%in the control group,（40.33±5.14）%in the 10μmol/L group,（22.07±0.31）%in the 20μmol/L group and（12.00±0.72）%in 30μmol/L group.The results of the cell scratch experiment showed that compared with the control group,the cell wound healing rate of different intervention time and different concentration of genistein group was significantly reduced（p<0.05）.The results of cell invasion experiments showed that compared with the control group,the number of cells passing through the cell in the different intervention time and different concentrations of genistein group was significantly reduced（p<0.05）.The cell cycle results showed that compared with the control group,the ratio of cells in the G₂/M phase of the 30μmol/L and 60μmol/L groups increased significantly.Apoptosis results showed that the 48 h and 72 h apoptotic rates in the 30μmol/L group were（31.96±10.17）%and（46.15±9.55）%respectively,and the 60μmol/L group cell apoptosis rates were（31.96±10.17）%and（46.15±9.55）%at 48 h and 72 h respectively.Compared with the control group,the apoptosis rate of them were significantly increased（p<0.05）.2.Through the experiment of subcutaneous transplantation tumor in nude mice,the tumor inhibition rate was（52.45±9.52）%,（48.04±12.96）%and（28.43±5.19）%after gavage with high,medium and low doses of genistein for21 days.2.Transcriptome sequencing revealed a total of 2 271 differential genes（p<0.05）,1 154 genes were up-regulated,1 117 genes were down-regulated,and 88 signal pathways were enriched by GO analysis.RT-qPCR results showed that the m RNA levels of 7 potential targets including CDK6,CYCLIN D,FGF2,STC2,P21,P53,AKT were significantly down-regulated（p<0.05）,and their relative expression levels were 0.37±0.06,0.37±0.06,0.3±0.19,0.23±0.05,0.69±0.09,0.12±0.03 and 0.38±0.02 times as compared to the control group,which are consistent with the sequencing results.Western blot experiments showed that after 30μmol/L Gen intervention,the protein expression levels of P21,AKT,STAT3,and PI3K in CNE1 cells were 3.18±0.1,1.42±0.56,1.07±0.45,0.1±0.04 times as compared to the control group,and significant changes occurred in the control group.Conclusion:1.Genistein can effectively inhibit the proliferation,colony formation,migration,and invasion of nasopharyngeal carcinoma CNE1 cells,block the nasopharyngeal carcinoma CNE1 cell cycle in G₂/M phase,and promote the apoptosis of CNE1 cells.2.Genistein can effectively inhibit the growth of nasopharyngeal carcinoma CNE1 cells in male nude mice.3.Genistein can effectively inhibit the growth of nasopharyngeal carcinoma CNE1 cells.Its anti-nasopharyngeal cancer mechanism may be inhibiting the expression of mutant P53 gene,restoring the function of wild-type P53 protein,and inhibiting the activity of PI3K/AKT pathway.