Helicobacter Pylori CagA Negatively Regulates Gastric Tumor Suppressor KLF4 Expression Through Ubiquitination Pathway

【Backgrounds】In the world,gastric cancer is the second leading cause of cancer death.The incidence of gastric cancer is different in gender,in general,the prevalence in men is twice as high as that in women,and the incidence of gastric cancer in different countries has a big difference.Overall,East Asia,Eastern Europe,South America and Central Europe,including China,have the highest incidence,while the lowest prevalence in most parts of Africa and North America.The differences in prevalence in some areas are mainly influenced by food storage,dietary structure,availability of fresh products and prevalence of Helicobacter pylori infection.Chronic Helicobacter pylori infection is the most risk factor for gastric cancer.About90% of new cases of non-cardiac gastric cancer worldwide are attributed to this bacterium,and WHO has long classified HP as the first biological carcinogen of gastric cancer.Cag A(cytotoxin-associated gene A)is an important virulence factor encoded by cag PAI(cag pathogenicity island)in Helicobacter pylori,and its carcinogenicity has been confirmed in animal models.Cag A,a bacterial oncoprotein,enters to cell via a specific T4 SS system and acts as a carcinogen in the cell.Cag A regulates intracellular signaling pathways by binding to multiple signaling proteins in the cells via phosphorylation or non-phosphorylation.On the other hand,Cag A can also inactivate a variety of intracellular tumor suppressor genes in the cells,such as Runx3,P53,and thus,plays a carcinogenic effect.Ubiquitination of proteasomal pathway is one of the most important ways of post-translational modification of proteins in the cell.It mainly regulates some short-lived and structurally abnormal proteins in the cell.Ubiquitination of proteins is a reversible process by the balance of processes of ubiquitination and deubiquitination.There are some specific deubiquitinating hydrolases in the body.Ubiquitination and de-ubiquitination together maintain normal cellular physiology and homeostasis.KLF4 is a zinc finger transcription factor that has a special PEST sequence located between the transcriptional activation domain and the transcriptional repression domain.Moreover,the half-life of KLF4 protein is short,and KLF4 plays a role of tumor suppressor gene in gastric cancer.In our previous experiments,the infection of Helicobacter pylori was simulated by transfecting the cag A plasmid in gastric epithelial cells or gastric cancer cells and Find that promoter methylation and altered mir-155 expression may be responsible for reduced KLF4 expression in gastric cancer,Down-regulate KLF4 expression from both transcriptional and translational levels to promote gastric cancer.Based on past research and the work of our laboratory,Therefore,we hypothesize that Cag A affects KLF4 expression at posttranslational levels through proteasome mediated ubiquitination pathway.What is the specific mechanism that causes degradation of ubiquitinated proteasome in KLF4? To test this hypothesis,we conducted the following experiments.【Research methods】1.Establishing a co-culture system of Helicobacter pylori and gastric epithelial cells.(1)First,we established Helicobacter pylori cell culture and identification methods.(2)GES-1 gastric epithelial cells were infected with HP,Establishing the co-culture system of Helicobacter pylori and gastric epithelial cells.2.Cag A affects KLF4 protein expression through the proteasome pathway.(1)The levels of KLF4 expression in gastric epithelial cells and gastric cancer cells were detected by WB;the half-life of KLF4 protein in GES-1 gastric epithelial cells was measured by WB.(2)GES-1 cells transfected with Cag A were treated with drugs CHX and MG-132,and then the half-life of KLF4 protein was measured according to the results of WB analysis.(3)GES-1 gastric epithelial cells and AGS gastric cancer cells were transfected with Cag A and /or KLF4 plasmids and then treated with MG-132 protease inhibitors.The changes of endogenous KLF4 protein and exogenous KLF4 protein were detected by WB.3.Cag A affects the expression of KLF4 by Ubiquitination pathway,which promotes the malignant transformation of cells.(1)The deubiquitinase U14,U21,U28,U42 and KLF4 plasmids were transfected into GES-1 cells and examined their effect on KLF4 protein expression by WB.(2)The expression of USP42 protein and RNA in gastric epithelial cells and several gastric cancer cells were detected by WB and PCR.(3)After HP infection or Cag A gene transduction,the expression of USP42 in GES-1 cells was detected by WB.(4)The effect of USP42 on KLF4 protein expression in GES-1 and AGS cells was examined by WB analysis after USP42 gene transduction.(5)The effects of USP42 knockdown on KLF4 epxression and cell behavioral changes in GES-1 cells were exiamsed after USP42 or KLF4 si RNA transfection.【Research results】1.We first established Helicobacter pylori cell culture and identification methods;GES-1 cells cultured with increased moi of Helicobacter pylori cells resulted in a dose-dependent downregulation of KLF4 protein expression,which is concomitant with increased Cag A protein expression in the cells.2.The levels of KLF4 expression are lower in gastric cancer cells than that in normal gastric epithelial GES-1 cells.3.KLF4 protein has a short half-life in GES-1 cell;the half-life of KLF4 protein in GES-1 cells transfected with Cag A is prolonged by MG132 treatment.4.MG-132 reversed the Cag A-induced down-regulation of endogenous and exogenous KLF4 protein.5.USP42 protein and RNA expression in gastric epithelial cells are higher than that in gastric cancer cells;Deubiquitinating enzyme USP42 can increase the expression of KLF4 in GES-1 cell.6.HP infection or Cag A gene transduction significantly reduced USP42 protein expression7.The AGS cells which have relatively low level of USP42 expression can upregulate KLF4 protein expression after transfected with USP42 expression vector;The GES-1 cells which have relative high level of USP42 expression can downregulated KLF4 protein expression after transfected with USP42 si RNA;The gastric epithelial cells that knocked down USP42 showed enhanced ability of proliferation,migration and clonogenicity.【Conclusions】1.HP infection or Cag A gene transduction can down-regulate the expression of KLF4 protein by the proteasome pathway.2.HP infection or Cag A gene transduction down-regulates the ubiquitination enzyme USP42 expression,whereas down-regulation of USP42 leads to down-regulation of KLF4 protein expression,suggesting that HP infection or Cag A gene transduction can negatively regulate the expression of KLF4 through the ubiquitination pathway,thereby promoting the malignant progression of the cells.