Mechanism Of Regulating KLF6 Protein Stability By Deubiquitinating Enzyme USP26

Objective There are few studies on the deubiquitination process of KLF6.This topic focuses on screening the deubiquitinating enzyme of KLF6,exploring the molecular mechanism of the deubiquitinating enzyme USP26 that to regulate KLF6 and its influence on the biological functions of cervical cancer cells.Methods(1)MG132 was added to Hela,MCF-7,Huh7 and other cell lines for 6 hours,Western blot detects the protein level of KLF6,and the cell lines with elevated KLF6 protein level were chosen as the target cell lines.(2)About 96 constructed deubiquitinase plasmids were transfected into 293 T and Hela cells to detect changes in KLF6 protein levels.(3)Overexpression and knockdown of USP26 to detect changes in the protein level of KLF6 in cells.(4)The cells were co-transfected with USP26 and KLF6,and treated with cycloheximide(CHX)at different time points.After the cells were collected,the changes in KLF6 protein levels were detected to determine whether USP26 could affect the attenuation rate of KLF6 protein.(5)Co-immunoprecipitation was used to detect the interaction between USP26 and KLF6,and to determine the interaction region between USP26 and KLF6.(6)The ubiquitination experiment explores the deubiquitination of KLF6 by USP26.(7)Clone formation,CCK-8,and scratch test were used to determine cell proliferation and migration ability,and to analyze the influence of USP26 on the biological behavior of cervical cancer cells.Results(1)Through the screening of the existing deubiquitinating enzyme library,the KLF6 deubiquitinating enzyme USP26 was found.(2)After USP26 was transfected in 293 T and Hela cell lines,the protein expression level of KLF6 was stabilized;after knocking down USP26,the protein expression level of KLF6 decreased.Half-life experiments have proved that USP26 can prolong the attenuation rate of KLF6 protein.(3)Co-immunoprecipitation experiments showed that there is an interaction between USP26 and KLF6,and it was found that the USP26 285-913 domain interacted with KLF6,but not the USP26 1-295 domain.(4)Wild-type USP26 can reduce the ubiquitination level of KLF6,while the USP26 enzyme mutant(USP26 C304S)loses its deubiquitination ability.(5)Biological experiments show that overexpression of USP26 can inhibit the proliferation and migration of Hela cells,and knockdown of USP26 can promote the proliferation and migration of Hela cells.ConclusionThe deubiquitinating enzyme USP26 can deubiquitinate KLF6,regulate the stability of KLF6,and is closely related to the proliferation and migration of cervical cancer cells.