| Objective:To investigate the effects of anlotinib combined with Tspan8 knockout on the proliferation,migration,invasion and apoptosis of colon cancer SW480 cells,and to explore the possible mechanism.Methods:1.The cancer tissues and normal tissues of patients who underwent surgical resection and were pathologically confirmed as colon cancer in our hospital were collected,and the expression of Tspan8 was detected by western blot.2.Western blot was used to detect the expression level of Tspan8 in colon cancer SW480,HCT116 and HT29,and the colon cancer cell lines with the highest expression of Tspan8 were selected for subsequent experiments.3.The gene sequence of Tspan8 was queried and copied in NCBI,and three single guide RNA（sg RNA）sequences were designed online using clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9（CRISPR/Cas9）technology,and the Tspan8 knockout plasmid was successfully constructed by ligating and recombining with Lenti-CRISPR V2 vector.Tspan8 lentivirus was successfully prepared by co-transfection of Tspan8 knockout plasmid into HEK293T tool cells by Lipofectamine TM2000.Colon cancer SW480 cells were infected with Tspan8 lentivirus,and the expression level of Tspan8 in colon cancer SW480 cells of each group was detected by western blot,and the cells with the best knockout effect were selected for subsequent experiments.4.MTT colorimetric assay was used to detect the proliferation of colon cancer SW480 cells treated with anlotinib at different concentrations（0,0.25,0.5,1,2,4,8,16,32,64 and 128μmol/L）for different times（24,48 and 72hours）,and half inhibitory concentration（IC50）was calculated to determine the subsequent experimental drug concentration.5.The experiment was divided into control group,anlotinib group,Tspan8knockout group and anlotinib+Tspan8 knockout group.MTT colorimetric assay was used to detect the proliferation ability of colon cancer SW480 cells in each group.Clonal formation assay was used to detect the clonogenesis ability of colon cancer SW480 cells in each group.Scratch repair test and Transwell chamber method were used to detect the migration ability and invasion ability of colon cancer SW480 cells in each group.Flow cytometry test was used to detect the apoptosis level of colon cancer SW480 cells in each group.6.Western blot was used to detect the effect of anlotinib on Tspan8 expression in colon cancer SW480 cells.Results:1.The results of Western blot showed that the expression level of Tspan8 in cancer tissues of colon cancer patients was significantly higher than that in normal tissues（p<0.01）.2.The results of Western blot showed that the expression level of Tspan8 in the three groups of colon cancer cells from high to low was as follows:SW480>HT29>HCT116.Compared with HT29 and HCT116 cells,the expression of Tspan8 in SW480 cells was the highest（p<0.05）.Therefore,the colon cancer SW480 cells were selected for subsequent experiments.3.The results of Positive cloning sequencing showed that three sg RNA sequences targeting Tspan8 gene were successfully and correctly inserted into lenti-CRISPR V2vector,indicating that the three Tspan8 knockout plasmids were successfully constructed.4.The results of Western blot showed that the expression level of Tspan8 in each group of colon cancer SW480 cells from high to low was as follows:SW480-WT>SW480-KO-Ⅰ>SW480KO-Ⅱ>SW480-KO-Ⅲ.Compared with SW480-WT,SW480-KO-Ⅰand SW480KO-Ⅱcells,the expression of Tspan8 in SW480-KO-Ⅲcells was significantly decreased（p<0.01）.Therefore,the colon cancer SW480-KO-Ⅲcells with the best knockout efficiency were selected for subsequent experiments.5.The results of MTT colorimetric assay showed that anlotinib could inhibit the proliferation ability of colon cancer SW480 cells at different concentration and different duration（p<0.01）,and the cellular proliferation inhibition rate increased significantly with the increase of anlotinib concentration and duration（p<0.01）.According to IC50,14μmol/L was selected as the subsequent experimental drug concentration.The results of cell proliferation experiment showed that compared with the control group,the proliferation ability of colon cancer SW480 cells was significantly inhibited in the anlotinib group,Tspan8 knockout group and anlotinib+Tspan8knockout group（p<0.01）,and the cell proliferation rate of the anlotinib+Tspan8knockout group was significantly lower than that of the anlotinib group at 2,3 and4days（p<0.01）,but there was no statistically significant difference between the two groups on 1day（p>0.05）,while the cell proliferation rate of the anlotinib+Tspan8knockout group was lower than that of the Tspan8 knockout group,and the difference was significant only on 1 and 2days（p<0.01）,but there was no statistically significant difference between the two groups on 3 and 4days（p>0.05）.6.The results of clonal formation assay showed that compared with the control group,the clonogenesis ability of colon cancer SW480 cells was significantly decreased in anlotinib group,Tspan8 knockout group and anlotinib+Tspan8 knockout group（p<0.01）,and the decrease level of cell cloning ability in the anlotinib+Tspan8knockout group was more significant than that in the anlotinib group（p<0.01）,while the anlotinib+Tspan8 knockout group had a lower level than the Tspan8 knockout group,but there was no statistical significance between the two groups（p>0.05）.7.The results of scratch repair test showed that compared with the control group,the migration ability of colon cancer SW480 cells was significantly decreased in the anlotinib group,Tspan8 knockout group and anlotinib+Tspan8 knockout group（p<0.01）,and the inhibition of cell migration in the anlotinib+Tspan8 knockout group was more significant than that in the anlotinib group and Tspan8 knockout group（p<0.01）.8.The results of Transwell chamber method showed that compared with the control group,the migration and invasion ability of colon cancer SW480 cells in the anlotinib group,Tspan8 knockout group and anlotinib+Tspan8 knockout group were significantly decreased（p<0.01）,and the inhibition of cell migration and invasion in the anlotinib+Tspan8 knockout group were more significant than that in the anlotinib group and Tspan8 knockout group（p<0.01）.9.The results of flow cytometry test showed that compared with the control group,the apoptosis level of colon cancer SW480 cells was significantly increased in the anlotinib group,Tspan8 knockout group and anlotinib+Tspan8 knockout group（p<0.01）,and the apoptosis level in the anlotinib+Tspan8 knockout group was higher than that in the anlotinib group and Tspan8 knockout group（p<0.01）.10.The results of Western blot showed that anlotinib could significantly decrease the expression of Tspan8 in SW480 cells of colon cancer（p<0.01）.Conclusion:Anlotinib combined with Tspan8 knockout could significantly inhibit the proliferation,migration and invasion of colon cancer SW480 cells and promote their apoptosis. |
PDF Full Text Request