Deubiquitinase USP6 Enhances Hela Cells Invasive Ability By Regulating C-Jun Protein Expression Levels

Objective As an important part of transcription factor complex AP-1,c-Jun plays an important role in the regulation of tumor differentiation and the induction of inflammation.Studies have shown that c-Jun is prone to be degraded by ubiquitination / proteasedependent pathways and is intracellularly unstable.Therefore,studies on the discovery of deubiquitinating enzymes(DUBs)which regulate the stability of c-Jun proteins is necessary.This article aims to search DUB expression libraries and explore ubiquitinases,which has the potential to modulate the stability of c-Jun proteins.After confirming the c-Jun ubiquitination-specific protease 6(USP6),we also observed the interaction mechanism between USP6 and c-jun protein and the effect of USP6 on the biological behavior of tumor cells.The mechanism of the downstream signaling of c-Jun was initially explored.Method(1)The library of deubiquitinase gene expression was constructed by molecular cloning technique,and all the plasmids in the library of deubiquitinase gene expression were transfected into 293 T cells by calcium phosphate transfection,the cells were lysed after 48 h and the proteins were extracted by Western Blot to detect deubiquitinase expression levels.(2)All the plasmids in the deubiquitinase gene expression library were transfected into Hela cells,and the cells were lysed after 48 h,the protein expression of c-Jun was detected by Western Blot.(3)We used the immunocoprecipitation assay to detect whether USP6 could directly ubiquitin c-Jun in vitro.(4)We constructed c-Jun knockdown stable cell line and used AP-1 reporter gene luciferase activity assay to detect the knockdown of c-Jun on USP6-induced AP-1 transcriptional activity.(5)The mRNA expression level of MMP10 was detected by Realtime-PCR,and the regulation of USP6 about the downstream gene MMP10 of c-Jun was analyzed.(6)The changes of cell invasion were detected by Transwell assay.Results(1)The library of deubiquitinase gene expression(a total of 68)was preliminarily constructed.The screening of deubiquitinase expression library showed that USP6 can regulate the stability of c-Jun protein.(2)In vitro ubiquitination experiments,wild-type USP6 can directly de-ubiquitinated c-Jun,USP6 active mutant c-Jun can not be directly de-ubiquitinated.(3)Knockdown c-jun can reduce the activity of the USP6 induced AP-1-phospholipase reporting gene.(4)Knockdown of USP6 can decrease the mRNA expression of MMP10 in Hela cells,and the knockdown of c-Jun can block the induction of MMP10 expression by USP6(5)Overexpression of USP6 could significantly enhance the invasiveness of Hela cells.The enhancement was suppressed by USP6 after knockdown of c-Jun.Knockdown of USP6 could inhibit the invasion ability of Hela cells.Conculsion The ubiquitination enzyme USP6 could stabilize c-Jun protein expression level,as well as enhance the AP-1 activity.USP6 could promote MMP10 transcription and enhance Hela cell invasion ability.