Activity And Mechanism Study Of Deubiquitination Specific Protease 8(USP8)on Proliferation,Migration And Invasion Of Gastric Cancer

BackgroundGastric cancer(GC)is a common malignant tumor and one of the major causes of cancer-related deaths globally.According to the global cancer statistics in 2020,there were 19.3 million new cancer cases and 10 million cancer-related deaths worldwide,and the incidence of gastric cancer ranked fifth.The incidence of gastric cancer was 5.6%and the mortality was 7.7%.Through more awareness,earlier detection and combined therapy,the survival of patients with GC has been improved to a certain extent.However,there are still some patients who will eventually die of recurrence and metastasis.Therefore,it is urgent to find new targeted genes and explore the specific transfer mechanism of GC.At present,the study of epigenetic inheritance in GC has been relatively sufficient.The most common epigenetic determinants include DNA methylation on cytosine residues,histone modification,non-coding RNA expression,chromatin structural remodeling,and nucleosome localization.Among them,histone modification mainly includes acetylation,methylation,phosphorylation,ubiquitination and so on.The ubiquitin-proteasome pathway is an important regulatory system for protein degradation in cells.Deubiquitination is a protein modification process catalyzed by deubiquitinating enzymes(DUBs).It has been reported that there are more than 100 DUBs encoded by human genes,which are divided into 6 subclasses according to their ubiquitin-protease domain.Ubiquitin-specific proteases(USPs)are the largest subclass of DUBs and ubiquitin-specific proteases 8(USP8)is a member of the USPs family.USP8 or ubitin isopeptidase Y(Ubipy),is a cysteine deubiquitination enzyme belonging to the USPs family.Currently,a large number of studies have reported that USP8 mutation can lead to the increase of epidermal growth factor receptor(EGFR)deubiquitination,which can down-regulate and maintain the EGF signaling pathway.The relationship between USP8 and cancer has also been extensively studied.The effect of USP8 on various tumors is different,and the high expression of USP8 in most tumors is associated with a poor prognosis.However,USP8 has only been studied in a small number of tumors,where there may be more valuable information.Understanding these changes may be very important for the diagnosis,treatment and prognosis of GC.This project aims to explore the mechanism of USP8 in the occurrence and development of GC.The research is carried out from the following four parts.Part I:Transcriptome and proteome sequencing analysis of target genes regulated by USP8 in GC.Part Ⅱ:Expression levels of USP8,HER-3 and HER-2 in cancer tissues of patients with GC and their relationship with prognosis of patients.Part Ⅲ:USP8 inhibitors inhibit the proliferation and invasion of HER-2 positive GC cells and its mechanism.Part Ⅳ:Down-regulation of USP8 inhibits proliferation,migration and invasion of HER-3 positive GC cells and its mechanism.Part I:Transcriptomics and proteomics analysis of USP8 target genes regulating gastric cancer cellsObjectiveTo explore the possible regulation of USP8 genes in GC cells and provide the theoretical and experimental basis for the regulatory mechanism of USP8 in GC.Methods1.The expression level of USP8 in NCI-N87,MKN-45,AGS,BGC-823,HGC-27 and MGC-803 GC cell lines were detected by western blot.2.The inhibitory rate of USP8 inhibitor on GC cell lines(IC50)was determined by MTT assay;3.The target genes regulated by USP8 on GC cells were detected by cell transcriptomics technology.4.The target proteins regulated by USP8 on GC cells were detected by cell proteomics technology.5.Correlation analysis of transcriptome sequencing results and proteome sequencing results and protein-protein STRING interaction analysis were performed.Results1.USP8 was highly expressed in NCI-N87,MKN-45,AGS,BGC-823,HGC-27 and MGC-803 GC cells;however,there was no significant difference in the expression of USP8 among different cells.2.GC cells MKN-45,NCI-N87,and AGS were significantly inhibited(all IC50 were<1 μmol/L),and MKN-45 was the most apparent(IC50=0.423 μmol/L).USP8 inhibitor had no significant inhibitory effect on MGC-803,HGC-27 and BGC-823 cells(all IC50>4μmol/L).3.A total of 1280 differential genes were detected by transcriptome sequencing,including 1014 up-regulated genes and 266 down-regulated genes.With a multiple changes greater than 2 times(up-regulated rate>2 or lower magnification<0.5,P<0.001),the total number of differentially expressed proteins between the inhibitor group and the normal control group was counted to 1001.Among them,792 proteins were up-regulated and 209 proteins were down-regulated.4.A total of 6098 proteins were identified by proteomics,of which 5922 proteins contained quantitative information.A total of 1280 differential proteins were detected,1014 were up-regulated and 266 were down-regulated.With a multiple change greater than 1.2 times(up-regulated rate>1.2 or lower magnification<1/1.2,P<0.001),the total number of differentially expressed proteins between the inhibitor group and the normal control group was 299.Among them,197 proteins were up-regulated and 102 proteins were down-regulated.5.In this study,31885 transcripts and 6098 proteins were quantified respectively through transcriptome and proteome quantitative studies.6034 genes were present at both transcriptome and proteome levels,and 1460 of them had different transcripts and proteins.There were 7 targets down-regulated and 17 targets up-regulated by both.STRING analysis showed that USP8 was directly correlated with BLM,HER-2 and HER-3,and HER-2 and HER-3 were positively correlated with USP8.The sequencing results of transcriptome and proteome of GC cells treated with USP8 inhibitors showed that HER-2 and HER-3 were significantly down-regulated(P<0.05).SummaryThis part revealed that the USP8 gene can target and regulate several GC genes,among which the target genes HER-2 and HER-3 are particularly prominent,but further studies are needed to verify.Part Ⅱ:Expression levels of USP8,HER-3 and HER-2 in cancer tissues of patients with gastric cancer and their relationship with prognosis of patientsObjective1.The expression level of USP8,HER-2 and HER-3 in paraffin specimens of GC patients were detected.2.To investigate the relationship between USP8,HER-2 and HER-3 and survival prognosis in patients with GC.Methods1.The expression level of USP8,HER-2 and HER-3 were detected by immunohistochemistry in 88 patients.Aperio Image Analysis software was used to analyze the immunohistochemical score.The Wilcoxon rank sum test was used to analyze the differences in the expression of USP8,HER-2 and HER-3 in cancer tissues and paired adjacent tissues.The expression differences of USP8,HER-3 and HER-2 in cancer tissues and paracancerous tissues were verified by GEPIA database.2.Relevant clinical data of patients with GC were collected.The correlation between the expression of USP8,HER-2 and HER-3 and clinical was analyzed by test.3.Kaplan-Meier method was used for survival analysis,and log-rank test was used to compare the expression level of USP8,HER-2 and HER-3 with the difference of survival rate.4.Pearson correlation analysis was used to determine the correlation between USP8 and the expression level of HER-2 and HER-3.5.Univariate and multivariate survival analysis of the prognosis of patients with GC was conducted by Cox proportional risk regression model to determine the independent influencing indexes of the prognosis of patients with GC.Results1.The expression values of USP8,HER-2 and HER-3 in cancer tissues were higher than those in paracancerous tissues(P<0.001).The GEPIA database analysis results also showed a relatively high expression trend of USP8,HER-2 and HER-3 in GC tissues.2.The high expression of USP8 in GC was correlated with regional lymph node metastasis and pTNM staging(P<0.05).The expression rate of USP8 was higher in patients with regional lymph node metastasis than in patients without lymph node metastasis.The expression of HER-2 in GC tissues was not correlated with age,sex,tumor size,tumor location,tumor differentiation degree,Lauren type,tumor invasion depth,regional lymph node metastasis,vascular tumor embolism,nerve invasion and pTNM stage(P>0.05).The high expression of HER-3 in GC tissues was correlated with nerve invasion,regional lymph node metastasis,and pTNM stage(P<0.05).3.Kaplan-Meier survival curves showed that patients with high expression of USP8,HER-2,and HER-3 showed significantly shorter survival times than those with low expression(P=0.009,0.001,and 0.018,respectively).USP8 combined with HER-2 and HER-3 detection in the prognosis of GC showed that,the survival rate of patients with low expression of HER-2 and USP8 was significantly higher than that of patients with high expression of HER-2 and USP8(P=0.006).Similarly,patients with low co-expression of HER-3 and USP8 had a better prognosis,while those with co-expression of HER-3 and USP8 had the worst prognosis(P=0.010).4.Pearson correlation analysis showed that there was a significant positive correlation between USP8 and HER-2 expression in GC tissues(R2=0.423,P<0.001),there was a significant positive correlation between USP8 and HER-3 expression in GC(R2=0.640,P<0.001).5.Analysis of clinicopathological indexes of 88 patients with GC by Cox risk proportional regression model.Male patients,Lauren hybrid,pTNM stage III,USP8,HER-2 and HER-3 high expression were affected the total survival and overall survival of the patients with GC.Those relative risks(RR)were 3.713,2.678,280.714,4.967,3.434 and 4.049,respectively.The multivariate comprehensive analysis results showed that the high expression of USP8,HER-3 and HER-2 could still be used as independent prognostic indicators of the GC patients.And those RR were 5.409,3.110 and 4.524,respectively.SummaryThe results of this part of clinical GC specimens showed that USP8,HER-2 and HER-3 could also be independent influencing factors for the prognosis of GC.Both USP8 and HER-3 and USP8 and HER-2 histones were positively correlated in GC,and the high co-expression of the two histones was associated with poor prognosis in GC patients.Part Ⅲ:USP8 inhibitors inhibit the proliferation and invasion of HER-2 positive gastric cancer cells and its mechanismObjectiveBased on the first and second parts,the role and mechanism of USP8 in regulating HER-2 and then affecting the progression of GC were further explored.Methods1.Western blot was used to detect the expression of HER-2 in GC cell lines.2.The IC50 of USP8 inhibitor against GC was detected by MTT.3.The effect of USP8 inhibitor on GC growth was detected by in vivo experiments.The expression of HER-3 and Ki-67 in xenograft tumors was detected by immunohistochemistry.4.Clonal formation assay and Transwell assay were used to detect the effect of USP8 inhibitor on proliferation and iInvasion of GC.5.Concentration gradient assay,half-life assay,immunofluorescence assay and immunoprecipitation assay were used to detect the mechanism of USP8 inhibitor regulating HER-2 in GC cells.Results1.In NCI-N87,MKN-45,AGS,BGC-823,HGC-27 and MGC-803 GC cell lines,HER-2 was highly expressed only in NCI-N87.2.The anti-proliferation effect of USP8 inhibitor on NCI-N87 was significantly higher than that of MGC-803(P<0.001),and IC50 was 0.78μmol/L(NCI-N87)and 12.40 μmol/L(MGC-803).3.The USP8 inhibitor significantly inhibited tumor growth in NCI-N87 cells,but failed to inhibit the growth of MGC-803 cells(P<0.05).Similarly,tumor volume in NCI-N87 mice was significantly inhibited over time compared with MGC-803(P<0.05).USP8 inhibitors prevented the growth of NCI-N87 xenograft tumors,but had no significant effect on body weight in mice inoculated with MGC-803 and NCI-N87(P<0.05).Immunohistochemical results of xenograft tumor showed that USP8 inhibitor significantly reduced the expression of HER-2 and Ki-67 in NCI-N87 compared with the control group(P<0.05),while there was no significant change in the expression of HER-2 and Ki-67 in xenograft tumor of MGC-803 mice(P>0.05).4.Clonal formation assay showed that USP8 inhibitors significantly inhibited proliferation of NCI-N87 cells compared with MGC(P<0.01).Transwell assay results showed that the invasion ability of NCI-N87 cells with inhibitor was reduced considerably(P<0.05),while MGC-803 cells were not affected.5.With the increase of USP8 inhibitor concentration,the expression of HER-2 decreased gradually,and the mRNA level of HER-2 decreased with the increase of USP8 inhibitor concentration.In NCI-N87 cells treated with USP8 inhibitor and si-RNA,Her-2 protein was almost completely degraded 6 h after treatment with CHX,compared with the control group.Immunofluorescence assay results showed that USP8 and HER-2 co-localized in NCI-N87 cells,while HER-2 fluorescence was not found in MGC-803.Ubiquitin fluorescence intensity was significantly increased and HER-2 decreased after USP8 inhibitor treatment,while MGC-803 did not change.The IP assay results of USP8 and HER-2 showed that USP8 could directly bind HER-2 in NCI-N87 cells,and the ubiquitination level of NCI-N87 cells was significantly increased after USP8 inhibitor treatment and si-RNA silencing treatment.SummaryUSP8 inhibitors can inhibit the proliferation and invasion of HER-2 positive GC cells,and USP8 may be a new potential therapeutic biomarker for HER-2 positive GC.Part Ⅳ:Down-regulation of USP8 inhibits proliferation,migration and invasion of HER-3 positive gastric cancer cells and its mechanismObjectiveBased on the first and second parts,the role and mechanism of down-regulation of USP8 in regulating HER-3 in GC progression were further explored.Methods1.Western blot was used to detect the expression of HER-3 in GC cell lines.2.Clonal formation assay was used to detect the effect of down-regulating USP8 on GC proliferation.3.Cell scratch and Transwell assay were used to detect the effect of down-regulation of USP8 on migration and invasion of GC.4.Concentration gradient test and half-life test were used to detect the effect of down-regulating USP8 on HER-3.5.Flow cytometry and apoptosis assay were used to detect the effect of down-regulating USP8 on HER-3 positive GC cells.6.Immunofluorescence and immunoprecipitation assay were used to detect the mechanism of down-regulation of HER-3 by USP8 in GC cells.7.The effect of down-regulated USP8 on GC growth was detected by animal experiments in vivo,and the expression of HER-3 and Ki-67 in xenograft tumor was detected by immunohistochemistry.8.Western blot was used to detect the association between down-regulated USP8 and PI3K/AKT signaling pathway in GC cells.Results1.In NCI-N87,MKN-45,AGS,BGC-823,HGC-27,and MGC-803 GC cell lines,HER-3 was highly expressed in NCI-N87,MKN-45,and AGS.2.Clonal formation assay results showed that there was no significant change between the USP8 inhibitor group and the control group or between the si-USP8 group and the control group in the MGC-803 cells that did not express HER-3.In AGS and MKN-45(HER-3 positive GC cells),the use of inhibitors and si-RNA significantly inhibited the proliferation of AGS and MKN-45(P<0.05).3.The effect of downregulation of USP8 on the migration of MGC-803 and MKN-45 cells was observed by cell scratch assay.We observed that the downregulation of USP8 had no significant effect on the migration of MGC-803 GC cells.Down-regulation of USP8 significantly inhibited the migration of AGS and MKN-45 GC cell lines.Transwell results showed that down-regulation of USP8 had no significant effect on the invasion ability of GC cell line MGC-803,while down-regulation of USP8 could significantly inhibit the invasion ability of AGS and MKN-45 GC cell line.4.With the increase of USP8 inhibitor concentration,the expression levels of HER-3 in NCI-N87,AGS and MKN-45 gradually decreased,respectively,and HER-3 was also significantly down-regulated si-USP8 group.Similarly,mRNA levels of HER-3 decreased with increasing USP8 inhibitor concentration.NCI-N87 and MKN-45 cells treated with USP8 inhibitors and si-RNA were treated with CHX,and the HER-3 protein in NCI-N87 and MKN-45 cells were almost completely degraded 6 h after treatment,compared with the control group.5.Down-regulation of USP8 could significantly inhibit the cell cycle of AGS and block its G1 phase,but MGC-803 was not affected.Down-regulation of USP8 significantly promoted the apoptosis of NCI-N87 and MKN-45 cells but did not affect MGC-803 cells.6.Immunofluorescence assay results showed that USP8 and HER-3 co-localized in MKN-45 and AGS cells,while no HER-3 fluorescence was found in MGC-803.Ubiquitin fluorescence intensity was significantly increased and HER-3 decreased after USP8 down-regulation,while MGC-803 did not change.IP assay results of USP8 and HER-3 showed that USP8 could directly bind HER-3 in NCI-N87 and MKN-45 GC cells.The ubiquitination level of HER-3 in NCI-N87 and MKN-45 GC cells was significantly increased after the USP8 inhibitor and si-RNA silencing treatment.7.There were no significant changes in tumor size,body weight and tumor weight in each group of MGC-803,while in the NCI-N87 and MKN-45 groups,tumor growth and tumor weight were significantly reduced in the inhibitor group compared with the blank control group.In addition,there was no significant effect on the growth of mice in each group.Immunohistochemical results of mouse tumors showed that the expressions of HER-3 and Ki-67 were significantly lower in the NCI-N87 and MKN-45 xenografted tumors than in the control group,while there were no significant changes in HER-3 and Ki-67 in the MGC-803 group.8.Downregulation of USP8 in NCI-N87 cells resulted in increased E-cadherin expression and decreased N-cadherin expression.The expression of p-AKT and p-PI3K in NCI-N87 was decreased after USP8 down-regulation,while no significant changes were observed in MGC-803 cells in the control group.SummaryDown-regulation of USP8 can degrade HER-3 in GC cells by ubiquitin,thereby inhibiting the proliferation,migration and invasion of GC cells.Conclusions1.USP8 gene can target several GC genes,among which the target genes HER-2 and HER-3 are particularly prominent.2.USP8,HER-2 and HER-3 can be used as independent prognostic factors of GC.Both USP8 and HER-3 and USP8 and HER-2 histones were positively correlated in GC,and the high co-expression of the two histones was associated with poor prognosis in GC patients.3.USP8 inhibitor can inhibit the proliferation and invasion of HER-2 positive GC cells.4.Down-regulation of USP8 can degrade HER-3 in GC cells by ubiquitin,thereby inhibiting the proliferation,migration and invasion of GC cells.