Effect Of Single Mutation Of Snail Protein Lysine Residue Site K98、K137 On LOXL2-mediated Snail And E-cadherin In Cholangiocarcinoma Cells

Objectives: 1.To study the effect of single mutation of Snail protein lysine residues K98 and K137 on LOXL2-mediated Snail protein and E-cadherin in cholangiocarcinoma cells.2.To provide partial experimental data for the development of targeted drugs for cholangiocarcinoma.Methods: In the RBE of recovered and passaged cholangiocarcinoma cells,the RBE was transiently transfected with EX-LOXL2 plasmid,EX-LOXL2 empty plasmid,EXLOXL2+Snail K98 S plasmid,EX-LOXL2+Snail K98 S empty plasmid,EXLOXL2+Snail K137 S plasmid,EX-LOXL2+Snail K137 S empty plasmid.The transfection effect of each group was detected by immunofluorescence technique.In RBE of cholangiocarcinoma cells transiently transfected with the corresponding plasmids,the changes of LOXL2 m RNA and Snail m RNA levels in each group after transfection were detected by q PCR.The binding of LOXL2 and Snail protein in each group was detected by CO-IP.The binding of LOXL2 and Snail protein in each group was detected by Western Blot.The expression of LOXL2,Snail protein and E-cadherin were detected by Western Blot.The changes of Snail protein in each group were detected by actinomycin treatment at 0h,1h,2h and 3h,respectively.By analyzing and comparing the changes of corresponding indexes in each group,the effects of single mutation of Snail protein lysine residue site K98 and K137 on LOXL2-mediated Snail protein and E-cadherin were clarified.Results:1.Cholangiocarcinoma cells were resuscitated,passaged,cultured with sufficient cells,and transiently transfected with the corresponding plasmids;the successful transient transfection of the corresponding plasmids was verified by immunofluorescence technique.2.LOXL2 m RNA and Snail m RNA expression in each group were detected by q PCR technique.Compared with the RBE group,the expression of LOXL2 m RNA and Snail m RNA in the EX-LOXL2 group was significantly higher(p<0.05);compared with the EX-LOXL2 group,the expression of LOXL2 m RNA and Snail m RNA in the EX-LOXL2+Snail K98 S and EX-LOXL2+Snail K137 S groups were not significantly.The expression of LOXL2,Snail protein and E-cadherin in each group was detected by Western Blot(p>0.05).Compared with the RBE group,the expression of LOXL2 and Snail protein were significantly higher in the EX-LOXL2group(p<0.05),and the expression of E-cadherin protein was significantly lower(p<0.05).Compared with EX-LOXL2 group,Snail protein expression was significantly decreased(p < 0.05),E-cadherin expression was significantly increased(p < 0.05)and LOXL2 expression was not significantly changed(p > 0.05)in EX-LOXL2+Snail K137 S group.Compared with EX-LOXL2 group,LOXL2,Snail protein and Ecadherin expression were not significantly changed in EX-LOXL2+Snail K98 S group(p>0.05).4.CO-IP results showed transient transfection of EX-LOXL2 plasmid,LOXL2 and Snail protein binding to each other,transient transfection of EX-LOXL2+Snail K98 S,EX-LOXL2+Snail K137 S plasmids,LOXL2 and Snail protein were bound to each other.5.Western Blot detected the expression of Snail protein in each group at 0h,1h,2h and 3h after actinomycin treatment.Compared with the RBE group,the Snail protein degradation rate was slowed down in the EX-LOXL2 group(p < 0.05);compared with the EX-LOXL2 group,the Snail protein degradation rate was accelerated in the EX-LOXL2+Snail K137 S group(p < 0.05);compared with the EXLOXL2 group,the EX-LOXL2+Snail K98 S group Snail protein degradation rate was not significantly changed compared with the EX-LOXL2 group(p > 0.05).Conclusions:1.EX-LOXL2 positively regulates LOXL2 and Snail protein and negatively regulates E-cadherin in wild-type cholangiocarcinoma cells,and slows down the rate of Snail protein degradation.2.Single mutation of Snail protein lysine residue site K98 and K137 may mediate the binding of LOXL2 to Snail protein.3.Single mutation of Snail protein lysine residue K137 negatively regulates LOXL2-mediated Snail protein in cholangiocarcinoma cells;positively regulates Ecadherin and accelerates the rate of Snail protein degradation.4.The Snail protein lysine residue site K137 site is expected to be a new target for cholangiocarcinoma treatment.