| Background and aims:Helicobacter pylori is a gram negative microaerobic bacterium that can parasitize the mucosal layer of human gastric tissue.Chronic Hp infection can induce the formation of the inflammatory immune microenvironment in gastric epithelial cells,trigger a Correa cascade reaction pattern,and ultimately develop into gastric cancer.Helicobacter pylori can induce the production of reactive oxygen species(ROS)in gastric epithelial cells,which is involved in promoting the occurrence of gastric cancer.At present,Hp infection is still the main pathogenic bacteria endangering human health.Studying the specific pathogenesis of Hp infection is of great significance for the prevention and treatment of Hp related diseases.SUMOylation modification is a three-level enzymatic cascade reaction which is similar to ubiquitination modification.SUMOylation modification regulates many biological processes,including DNA damage repair,immune reactions,carcinogenesis,cell cycle progression,and apoptosis.Many studies have found that enzymes related to the SUMO modification pathway are highly expressed in various human malignant tumors,and SUMO molecules can bind to a variety of cancer promoting proteins to enhance their cancer promoting effects.The imbalance of SUMO modification is closely related to the development of tumors.SAE1 is a subunit of E1 activating enzyme during the SUMO modification process.Some studies have shown that SAE1 is highly expressed in human glioma and promotes the progression of human glioma.SAE1 is considered as a promising biomarker for diagnosing cancer metabolism in hepatocellular carcinoma.The relationship among SAE1 and SUMO modification and gastric cancer is not very clear.The homeostasis of SUMO modification is influenced by many environmental and pathological factors.Some studies have shown that bacterial pathogens can intervene through the SUMO modification pathway of the host to ensure successful infection.Different bacterial pathogens have different targeted interventions during the SUMOylation modification process of the host.These interventions have led to changes in the proteome and transcriptome of host cells,and have indirectly altered normal signal transduction in host cells.As a common gastric pathogen,the relationship between Hp and SUMO modification is currently unclear.Helicobacter pylori can induce the production of reactive oxygen species(ROS)in gastric epithelial cells.It has been reported that the level of reactive oxygen species in cells can affect the SUMOylation modification state,and whether Hp can affect the SUMOylation modification level of cells through the ROS pathway also requires further exploration.Based on the above research background,this study explored the relationship among SAE1 and SUMO modification and Hp infection from the molecular,cellular,and tissue levels,as well as the mechanism in Hp related gastric cancer,so as to providing more theoretical basis for the prevention and treatment of Hp related diseases and strategy of targeting SUMO modification process for tumor treatment.Materials and Methods:I.Expression of SUMO molecules and enzymes involved in SUMO modification pathway in gastric cancer tissue.1.TIMER database analysis showed that the m RNA expression of SUMO molecule and SUMO modified E1 enzyme SAE1/SAE2,E2 enzyme UBC9 in various human malignant tumors was higher than that in normal tissues(P<0.05).2.GEPIA database analysis showed that SUMO1(P<0.05),SUMO2(P<0.05),and SUMO-modified E1 enzyme SAE1/SAE2,E2 enzyme UBC9(P<0.05)m RNA expression in gastric cancer was higher than that in normal tissues;3.Immunohistochemical results showed that the expression of SUMO molecules,SUMO modified E1 enzymes SAE1/SAE2,and E2 enzyme UBC9 in gastric cancer was higher than that in normal gastric tissue(P<0.05).II.Effects of Hp infection on the expression of SUMO molecules and enzymes involved in the SUMO modification pathway.1.The Hp strain PMSS1 was co cultured with GES-1 and AGS cells at different infection multiples and infection duration.Western blot showed that Hp infection significantly upregulated the expression of SUMO1,SUMO2/3,and SAE1(P<0.05),but had no significant effect on the expression of SAE2,UBC9.2.Western blot showed that NAC could significantly inhibit the expression of SUMO1,SUMO2/3,and SAE1 in GES-1 and AGS cells treated with different concentrations of ROS inhibitor NAC and different durations(P<0.05).3.Western blot showed that hydrogen peroxide significantly upregulated the expression of SUMO1,SUMO2/3,and SAE1 after stimulating reactive oxygen species in cells treated with different concentrations of hydrogen peroxide(P<0.05).4.Set negative and positive controls of ROS,pure Hp infection group,Hp infection and NAC combined treatment group,and detect the ROS level of each group with the ROS detection kit.Hp infection can significantly increase the ROS level of cells(P<0.05),hydrogen peroxide solution can significantly stimulate ROS in cells(P<0.05),and NAC can significantly inhibit the ROS produced by Hp induced cells(P<0.05).Western blot showed that Hp infection significantly increases the SUMO1,SUMO2/3,and SAE1 expression.The expression of SUMO1,SUMO2/3 and SAE1 was significantly inhibited by NAC(P<0.05).NAC significantly inhibited the upregulation of SUMO1,SUMO2/3,and SAE1 induced by Hp(P<0.05).III.Effects of SAE1 on the biological behavior of gastric epithelial cells.1.Constructed the small interfering RNA to knockout SAE1 in AGS cells.Western blot showed that si-SAE1 significantly knockdown the expression of SAE1 in AGS cells(P<0.05)2.After transfection of si-SAE1 into AGS cells,plate cloning experiments showed that the proliferation ability of cells in the si-SAE1 group was weaker than that in the control group(P<0.05).3.After transfection of si-SAE1 into AGS cells,Transwell cell migration and invasion experiments showed that the migration and invasion abilities of si-SAE1 group cells were weaker than those of the control group cells(P<0.05).4.After transfection of si-SAE1 into AGS cells,Western blot showed a decrease in the expression of gastric epithelial mesenchymal transformation(EMT)related proteins(P<0.05).IV.The role of SAE1 in Hp infection-induced biological behavior of gastric epithelial cells.1.After transfecting AGS cells with si-SAE1 and infecting cells with Hp strain PMSS1,the CCK8 experiment showed that si-SAE1 could significantly inhibit the ability of Hp to promote the proliferation of gastric epithelial cells(P<0.05).2.After transfection of si-SAE1 into AGS cells,the plate cloning experiment showed that si-SAE1 could significantly inhibit the ability of Hp to promote the proliferation of gastric epithelial cells(P<0.05).3.After transfection of si-SAE1 into AGS cells,the results of Transwell cell migration and invasion experiments showed that si-SAE1 could significantly inhibit the ability of Hp to promote the migration and invasion of gastric epithelial cells(P<0.05).4.After transfecting AGS cells with si-SAE1,the whole cell lysates of each group of cells were extracted.Western blot results showed that si-SAE1 could significantly inhibit the upregulation of EMT-related indicators promoted by Hp in gastric epithelial cells(P<0.05).V Relationship between SAE1 expression in gastric cancer tissues and clinical information of patients.1.TCGA database analysis showed that SAE1 m RNA was highly expressed in gastric cancer compared to normal gastric tissue(P=0.001),and the overexpression of SAE1 was associated with poor prognosis in patients.2.Immunohistochemical results showed that SAE1 was highly expressed in gastric cancer tissue microarray compared to normal adjacent tissues.Statistical analysis of the correlation between SAE1 expression in gastric cancer tissue microarray and clinical information of patients showed that SAE1 was highly expressed in gastric cancer tissue(P<0.0001),and the high expression of SAE1 was significantly correlated with TNM stage(P=0.029)and vascular invasion(P=0.029)of patients,The overall survival time of the SAE1 high expression group was worse than that of the SAE1 low expression group.Results:I.Expression of SUMO molecules and enzymes involved in SUMO modification pathway in gastric cancer tissue.1.TIMER database analysis showed that the m RNA expression of SUMO molecule and SUMO modified E1 enzyme SAE1/SAE2,E2 enzyme UBC9 in various human malignant tumors was higher than that in normal tissues(P<0.05);2.GEPIA database analysis showed that SUMO1(P<0.05),SUMO2(P<0.05),and SUMO-modified E1 enzyme SAE1/SAE2,E2 enzyme UBC9(P<0.05)m RNA expression in gastric cancer was higher than that in normal tissues;3.Immunohistochemical results showed that the expression of SUMO molecules,SUMO modified E1 enzymes SAE1 /SAE2,and E2 enzyme UBC9 in gastric cancer was higher than that in normal gastric tissue(P<0.05).II.Effects of Hp infection on the expression of SUMO molecules and enzymes involved in the SUMO modification pathway.1.The Hp strain PMSS1 was co cultured with GES-1 and AGS cells at different infection multiples and infection duration.Western blot showed that Hp infection significantly upregulated the expression of SUMO1,SUMO2/3,and SAE1(P<0.05),but had no significant effect on the expression of SAE2,UBC9.2.Western blot showed that NAC could significantly inhibit the expression of SUMO1,SUMO2/3,and SAE1 in GES-1 and AGS cells treated with different concentrations of ROS inhibitor NAC and different durations(P<0.05).3.Western blot showed that hydrogen peroxide significantly upregulated the expression of SUMO1,SUMO2/3,and SAE1 after stimulating reactive oxygen species in cells treated with different concentrations of hydrogen peroxide(P<0.05).4.Set negative and positive controls,pure Hp infection group,Hp infection and NAC combined treatment group,and detect the ROS level of each group with the ROS detection kit.Hp infection can significantly increase the ROS level of cells(P<0.05),hydrogen peroxide solution can significantly stimulate ROS in cells(P<0.05),and NAC can significantly inhibit the ROS produced by Hp induced cells(P<0.05).Western blot showed that Hp infection significantly increases the SUMO1 The expression of SUMO2/3 and SAE1 was significantly inhibited by NAC(P<0.05).NAC significantly inhibited the upregulation of SUMO1,SUMO2/3,and SAE1 induced by Hp(P<0.05).III.Effects of SAE1 on the biological behavior of gastric epithelial cells.1.Construction of small interfering RNA from knockout cell SAE1.Western blot showed that si-SAE1 significantly knockdown the expression of SAE1 in AGS cells(P.2.After transfection of si-SAE1 into AGS cells,plate cloning experiments showed that the proliferation ability of cells in the si-SAE1 group was weaker than that in the control group(P<0.05).3.After transfection of si-SAE1 into AGS cells,Transwell cell migration and invasion experiments showed that the migration and invasion abilities of si-SAE1 group cells were weaker than those of the control group cells(P<0.05).4.After transfection of si-SAE1 into AGS cells,Western blot showed a decrease in the expression of gastric epithelial mesenchymal transformation(EMT)related proteins(P.IV.The role of SAE1 in Hp infection-induced biological behavior of gastric epithelial cells.1.After transfecting AGS cells with si-SAE1 and infecting cells with Hp strain PMSS1,the CCK8 experiment showed that si-SAE1 could significantly inhibit the ability of Hp to promote the proliferation of gastric epithelial cells(P<0.05).2.After transfection of si-SAE1 into AGS cells,the plate cloning experiment showed that si-SAE1 could significantly inhibit the ability of Hp to promote the proliferation of gastric epithelial cells(P<0.05).3.After transfection of si-SAE1 into AGS cells,the results of Transwell cell migration and invasion experiments showed that si-SAE1 could significantly inhibit the ability of Hp to promote the migration and invasion of gastric epithelial cells(P<0.05).4.After transfecting AGS cells with si-SAE1,the whole cell lysates of each group of cells were extracted.Western blot results showed that si-SAE1 could significantly inhibit the upregulation of EMT-related indicators promoted by Hp in gastric epithelial cells(P<0.05).V Relationship between the expression of SAE1 in gastric cancer tissues and clinical information of patients.1.TCGA database analysis showed that SAE1 m RNA was highly expressed in gastric cancer compared to normal gastric tissue(P=0.001),and the overexpression of SAE1 was associated with poor prognosis in patients.2.Immunohistochemical results showed that SAE1 was highly expressed in gastric cancer tissue microarray compared to normal adjacent tissues.Statistical analysis of the correlation between SAE1 expression in gastric cancer tissue microarray and clinical information of patients showed that SAE1 was highly expressed in gastric cancer tissue(P<0.0001),and the high expression of SAE1 was significantly correlated with TNM stage(P=0.029)and vascular invasion(P=0.029)of patients,The overall survival time of the SAE1 high expression group was worse than that of the SAE1 low expression group.Conclusions:1.SUMO molecule and E1 enzyme SAE1/SAE2 of SUMO modification pathway,and E2 enzyme UBC9 are highly expressed in gastric cancer tissue.2.Hp infection can upregulate the expression of SAE1,SUMO1,and SUMO2/3in gastric epithelial cells through the ROS pathway,but has no significant effect on the expression of SAE2,UBC9.3.Knocking down the expression of SAE1 in gastric epithelial cells can significantly inhibit the proliferation,migration,invasion ability,and EMT phenotype of gastric epithelial cells promoted by Hp.4.SAE1 is highly expressed in human gastric cancer tissue and is associated with poor prognosis in patients. |
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