Sphase Kinase-associated Protein 1 Is Associated With Proliferation And Invasion Of Intrahepatic Cholangiocarcinoma Cells

ObjectiveTo investigate the expression of S-phase kinase-associated protein 1(SKP1),a core component of ubiquitin linker E3,in the liver bile duct cell carcinoma tissue and adjacent normal bile duct tissue.In vitro experiments regulate the expression of SKP1,and further clarify the effects of SKP1 on the proliferation,migration and invasion of intrahepatic bile duct cells(RBE).MethodTwenty-five cases of intrahepatic cholangiocarcinoma tissues and adjacent normal bile duct tissues of about 2 cm who underwent radical resection at the People’s Hospital of Henan University from August 2017 to April 2019.Collect certain quality specimens,put them into cryopreservation tubes,transfer and save liquid nitrogen,and make detailed registration.This study was approved by the Ethics Committee of Henan University People’s Hospital(Henan Provincial People’s Hospital),and all patients signed the informed consent form for specimen collection.Culture human hepatic bile duct cell carcinoma cells(RBE)and human embryonic kidney cells(293T),and master the cell passage rules.The gene library was used to design and construct the SKP1 expression interference vector and blank vector.The lentiviral packaging technology was used to stably transfect into the intrahepatic bile duct cancer cells.The antibiotics were selected to form the experimental group and the control group.The protein lysate was used to extract tissue protein and cellular protein,and to determine the protein concentration.Western-Blot technology was used to detect the expression difference between SKP1 clinical specimens and stably transfected cells.Statistical analysis was performed to draw conclusions.When the experimental group and the control group are successfully constructed.CCK8 measured absorbance and calculated the average relative survival rate of the two groups to obtain the effect of SKP1 on RBE’s proliferation ability;the average number of cells crossed in the scratch test to reflect the effect of SKP1 on RBE migration ability;the Transwell chamber experiment counted through the small hole The number of cells stained with purple indirectly reflects the effect of SKP1 on the invasion ability of RBE.In the end,all data were analyzed statistically using SPSS22.0.The results were expressed as mean ± standard deviation(Mean ± SD).The data between the two groups were tested by t test.The difference was statistically significant with p <0.05.Results1.In clinical tissue samples,SKP1 expression in intrahepatic cholangiocarcinoma tissue was(1.701 ± 0.215)times that of normal bile duct tissue,and the difference was statistically significant(t = 8.512 p <0.05).2.The experimental group and control group were successfully constructed by transfecting RBE cells with lentivirus,and the expression of SKP1 in the experimental group was reduced.According to Western-Blot test,the expression of SKP1 in the experimental group was reduced(3.632 ± 0.335)times compared with the blank group.The difference was statistically significant(t = 11.376 p <0.01).3.The absorbance(A value)of the experimental group measured by CCK8 was 0.316 L /(g · cm)at 24 hours,0.524 L /(g · cm)at 48 hours,and 0.527 L /(g · cm)at 72 hours.The average absorbance of the control group was 0.594 L /(g · cm)for 24 hours,1.228 L /(g · cm)for 48 hours,and 1.503 L /(g · cm)for 72 hours.The average relative survival rates of the experimental group and the control group were(53.136 ± 0.314)%,(42.661 ± 0.248)%,and(35.037 ± 0.203)%,and the differences were statistically significant(t 8.585 8.899 9.210 p <0.05).4.The scratch test showed that the average number of migrated cells in the experimental group at 24 and 48 hours was(41.198 ± 3.092)and(64.773 ± 6.437)compared with the control group(113.487 ± 3.514)and(168.767 ± 8.151).The differences were statistically significant.Significance of scientific analysis(t was 5.430 6.224 p <0.05).5.The results of Transwell experiment showed that the average number of cells passing through the foramen in the experimental group was(34.185 ± 3.124)significantly lower than that in the control group(121.234 ± 6.814),and the difference was statistically significant(t = 9.918 p <0.05).Conclusion1.The expression of SKP1 in cholangiocarcinoma of liver is higher than that of normal bile duct;2.Decreasing the expression of SKP1 can effectively inhibit the proliferation,migration and invasion of intrahepatic cholangiocarcinoma.