The Role And Mechanism Of Snail Site In The Regulation Of E-Cadherin By LOXL2 In Cholangiocarcinoma Cells

Objective(s):In the RBE cell line of cholangiocarcinoma,the study of LOXL2 mediated Snail and its site provides a new therapeutic basis for the targeted treatment of cholangiocarcinoma,and also provides a new therapeutic idea for other tumors.Methods:The wild type was established by transient transfection of K98/K137 double mutant plasmid,EX-LOXL2 plasmid and LOXL2-Sh RNA interfering plasmid in cholangiocarcinoma RBE cells,EX-LOXL2-MU-S-k98-k137 Group,EX-LOXL2 group,Sh-LOXL2-MU-S-k98-k137 group were compared with Sh-LOXL2 group and corresponding empty plasmid group.The amount of LOXL2 and Snail m RNA and LOXL2 in human cholangiocarcinoma RBE cells were detected by q-PCR and Western Bolt,the half-life of Snail and E-cadherin was measured by cycloheximide assay.Results:Using q-PCR,Western Bolt,and actinomycin assay methods to detect RBE in cholangiocarcinoma cells at the gene and protein levels,the following results can be obtained.1.Instantaneous transfection of EX-LOXL2 plasmid and LOXL2-Sh RNA interference plasmid in cholangiocarcinoma cell RBE.The results of q-PCR and Western Blot experiments showed statistical differences in the expression of LOXL2 m RNA and protein between the two groups,confirming the successful transfection of the plasmid.The q-PCR experiment detected the relationship between LOXL2 and Snail at the m RNA level.Compared with RBE,the EX-LOXL2 group showed a significant increase in Snail m RNA expression,P<0.001.The expression of Snail m RNA was significantly increased in the Sh-LOXL2 group,P<0.001,and the amount of Snail m RNA was significantly reduced in the Sh-LOXL2 group compared to the EX-LOXL2 group,P<0.001.Western Blot assay was used to detect the relationship between LOXL2 and Snail at protein expression levels.Compared to RBE,the expression of Snail protein increased in the EX-LOXL2 group,P<0.001,while decreased in the ShLOXL2 group,P<0.05;The EX-LOXL2 group showed an increase in Snail protein expression compared to the Sh-LOXL2 group,P<0.001.2.In RBE cells of cholangiocarcinoma,q-PCR and Western Blot experiments were used to detect the expression of Snail m RNA and protein levels at the k98/k137 double mutation Snail site.Compared with the EX-LOXL2 group and the EX-LOXL2MU-k98-k137 group,there was no statistically significant difference in LOXL2 m RNA levels between the Sh-LOXL2 group and the Sh-LOXL2 MU-k98-k137 group,p>0.05.There was no statistically significant difference in LOXL2 protein content between the EX-LOXL2-MU-S-k98-k137 group and the EX-LOXL2 group,and between the Sh-LOXL2-MU-S-k98-k137 group and the Sh-LOXL2 group,p>0.05.The EX-LOXL2-MU-S-k98-k137 group showed a significant decrease in Snail m RNA expression compared to the EX-LOXL2 group,p<0.0001;There was no statistical difference between the Sh-LOXL2-MU-S-k98-k137 group and the Sh-LOXL2 group,P>0.05.Compared with the EX-LOXL2-MU-S-k98-k137 group,the EX-LOXL2 group showed an increase in Snail protein content,P<0.01;There was no statistical difference between the Sh-LOXL2-MU-S-k98-k137 group and the Sh-LOXL2 group,P>0.05.3.In the RBE of cholangiocarcinoma cells,the half life of Snail was measured using actinomycin assay.After 3 hours,the content of Snail degrading protein in the EX-LOXL2-MU-S-k98-k137 group was significantly lower than that in the EXLOXL2 group,P<0.01.Compared to the EX-LOXL2 group,the protein content of Snail after degradation in the RBE group and Sh-LOXL2 group significantly increased,P<0.01.4.Western Blot assay was used to detect the expression of E-cadherin protein in RBE cells of cholangiocarcinoma.Compared with RBE,the expression of E-cadherin protein increased in the Sh-LOXL2 group,P<0.05,while it decreased significantly in the EX-LOXL2 group,P<0.05;The protein content in the EX-LOXL2 group was significantly reduced compared to the Sh-LOXL2 group,P<0.001.The EX-LOXL2 group showed a decrease in E-cadherin protein content compared to the EX-LOXL2MU-k98-k137 group,and the Sh-LOXL2 group showed a decrease in E-cadherin protein content compared to the Sh-LOXL2 MU-k98-k137 group,P<0.05.Conclusion(s):1.In RBE cells of cholangiocarcinoma,LOXL2 can positively regulate Snail m RNA and protein expression,improve the stability of Snail,and the interaction between LOXL2 and Snail jointly downregulates the protein expression level of Ecadherin.2.In RBE cells of cholangiocarcinoma,the Snail site k98/k137 double mutation can downregulate the expression levels of Snail m RNA and protein,weaken Snail stability,increase degradation rate,and then upregulate the expression of E-cadherin protein.