Study On The Mechanism Of Deubiquitination Enzyme Usp8 Regulating Cell Migration By Activating JNK Signaling Pathway

Cancer,also known as malignant tumors,has become the leading cause of death worldwide.Tumor metastasis,rather than primary tumor overgrowth,is the main cause of tumor-induced death.Metastasis of malignant tumor is a complex process controlled by many genes,and its underlying mechanism is still unclear.Therefore,it is very useful to explore new regulatory factors related to tumor cell migration to provide methods for cancer treatment.In the past for more than half a century,Drosophila melanogaster since it has short life cycle,easy feeding,fecundity,chromosome,mutant many advantages,such as,it has become a very important research on tumor cell migration model of biology.Studies show that the migration of tumor cells is a complex mechanism and by multiple genes and signaling pathways involved in the process.The wing disc of Drosophila became an ideal model for studying tumor cell migration in Drosophila.More and more to Drosophila to the research of cell migration model show that JNK signaling pathway not only has great relationship with cell apoptosis,and it can control the movement and adhesion cells in the process of development.The activation of JNK signaling pathway can result in tumor cell migration.So the JNK signaling pathway play an important role in the process of the migration of tumor cells.Deubiquitination enzyme 8（Usp8）encodes a protease involved in protein breakdown.It contributes to the integrity of the ESCRT sorting machinery and the regulation of Hedgehog and Wingless signaling pathways.Usp8 is involved in several processes,including neuronal remodeling,positive regulation of receptor recirculation,and positive regulation of signal transduction.Usp8 is located in the cytoplasm and is expressed in the head and eyes of adult Drosophila.The direct human homolog of this gene is associated with ACTH-secreting pituitary adenomas.Homology with human USP8（deubiquitination enzyme）.However,up to now,the role and mechanism of the deubiquitination enzyme Usp8 in tumor cell migration and motility is not clear.In this study,deubiquitination enzyme Usp8 was used as the research object in Drosophila.Expression vector construction,microinjection technology,specific tissue expression system,immunofluorescence antibody staining and confocal,cell culture,transfection,immunoprecipitation and western blot,real-time fluorescence quantitative PCR,mouse antibody preparation,si RNA,GST pull-down and statistical analysis were used to explore the effect of Usp8 on tumor cell migration and further analyze its regulatory mechanism.The main experimental results are as follows:（1）Usp8 was overexpressed by transgenic technology,FLP-out technology and GAL4system.It was found that overexpression of usp8 could activate cell migration.Subsequently we overexpressed or knockdowned usp8 in the context of scrib RNAi interference.Historically,scrib RNAi does trigger invasive cell migration,which is attenuated by usp8knockdown and exacerbated by usp8 overexpression,suggesting that Usp8 promotes cell migration.（2）By immunofluorescence,we found that inhibition of endogenous JNK signaling by Bsk^DN effectively inhibited enhanced tumor cell migration induced by usp8 expression compared to usp8 alone.Meanwhile,the expression levels of puc-lac Z,Mmp1 and p JNK were significantly increased.Puc,mmp1 and jnk were classic target genes of JNK signaling pathway.This suggests support for Usp8 activation of JNK pathway to promote tumor cell migration.（3）Genetic upper-level experiments showed that overexpression of usp8 induced upregulation of puc-lac Z could be restored by bsk RNAi,hep RNAi or tak1 RNAi,but not by traf2 RNAi,suggesting that Usp8 was located downstream of Traf2,but Tak1 upstream regulated JNK pathway.Co-IP results showed that Usp8 and Tak1 interacted with each other whether endogenous or exogenous.Co-IP and GST pull-down analysis showed that both N-terminal and C-terminal of Usp8 could bind Tak1,while Tak1-N could bind Usp8 and Tak1-C could not.（4）Through stability and ubiquitination experiments,we found that Tak1 has a half-life of 3 hours.Usp8 stabilizes Tak1 in a dose-dependent manner and Usp8 stabilizes Tak1 by reducing the multiubiquitination of K48 and K63 on Tak1.（5）Since the key components and regulatory mechanisms of JNK pathway are conserved in Drosophila and mammals,we conducted conserved experiments and found that human USP8 can replace d Usp8 in Drosophila wing disc to activate the expression of puc-lac Z,increase the protein level of Mmp1,and promote the migration of tumor cells.And the migration of such tumor cells can be attenuated by bsk^DN or tak1 RNAi.Together,these results suggest that hUSP8 can replace d Usp8 to trigger cell migration in a Tak1-dependent manner.（6）Co-IP analysis showed that hUSP8 interacts with hTAK1.hUSP8 knockdown reduces TAK1 protein,while hUSP8 overexpression has the opposite effect.Analysis of breast cancer samples showed that hUSP8 protein expression level was positively correlated withTAK1.Fg-hUSP8 overexpression was elevated,while hUSP8 knockdown reduced MMP9（homologue of human Mmp1）and p JNK levels.q RT-PCR showed that overexpression of hUSP8 promoted the transcription of target genes of JNK pathway,including MMP9 and DUSP10（homologue of human puc）.These results suggested that hUSP8 activated JNK signaling pathway activity by stabilizing hTAK1 protein.At the same time,Transwell analysis showed that hUSP8 significantly promoted tumor cell migration,while knockdown of hUSP8 inhibited tumor cell migration.In summary,our results suggest that the Usp8-Tak1 module promotes tumor cell migration,and further suggest that this module fine-regulates JNK signaling pathway activity at the level of promoting tumor cell migration and post-translational modification with high conserved nature.This study not only further enables us to understand the biological function of deubiquitination enzyme,but also plays a significant role in promoting the development of breast cancer research and provides a new theoretical basis and research direction for the clinical treatment of cancer.