Effect Of Helicobacter Pylori CagA Protein On Energy Metabolism Of Gastric Cancer Cells

Objective:To investigate the effect of Helicobacter pylori（H.pylori）Cytotoxin related gene A protein（Cag A）on energy metabolism of gastric cancer cells,establish a Cag A-induced energy metabolism network for gastric cancer cells,and explore the molecular mechanism of Cag A promoting gastric cancer development from the perspective of energy metabolism.Methods:（1）Establish a gastric cancer cell model that overexpresses cag A.Construct and identify the recombinant adenovirus vector p Adeno-cag A over-expressing cag A,infect primary gastric cells WZK and YHD with adenovirus,and observe the morphological changes of the cells.Western blot（WB）and Immunofluorescence（IF）was used to detect the expression and cell localization of Cag A protein.（2）The effect of overexpression of Cag A on cellular glycolytic enzymes and GLUT1.（1）Detection of glycolytic enzymes such as hexokinase 2（HK2）,enolase-α（ENO1）,pyruvate kinase 1/2（PKM1/2）,lactate dehydrogenase A（LDHA）and glucose transporter 1（GLUT1）Protein in adenovirus-infected WZK and YHD cells.（2）Lipofectamine 2000 was used to transfect gastric cancer cells by overexpressing glut1 plasmid p CDH-glut1 and empty plasmid,and screened by puromycin.BGC823/glut1 and AGS/glut1 stable cell lines were verified by WB and IF.IF was used to detect the co-localization of GLUT1 and Cag A.（3）The effect of overexpression Cag A on the relevant indicators of cell energy metabolism.AGS、YHD、BGC-823/glut1 and AGS/glut1 gastric cancer cells infected with adenovirus were divided into blank group（Mock）,empty adenovirus group（NC）and Cag A adenovirus group（Cag A）.（1）The ATP concentration,glucose uptake and glucose consumption were detected by multi-functional microplate reader.（2）The ATP rate and mitochondrial function were detected in p Adeno-cag A-infected AGS and YHD cells by Seahorse Energy Metabolic Analyzer.（4）Metabolomics research.（1）Relative quantitative energy metabolism:Adenovirus-infected YHD 72 h cell samples were collected and divided into NC group and Cag A group,each group of 5 biological replicates,using liquid chromatography-tandem mass spectrometry（LC-MS/MS）analysis method,targeting 29 energy metabolites Perform relative quantitative analysis.（2）Analysis of glucose metabolic flux based on ¹³C labeling:AGS cells were infected with p Adeno-cag A for 30 h and replaced with high glucose DMEM containing ¹³C labeled glucose,divided into NC group and Cag A group.Each group had 5 biological replicates,and isotope metabolites were analyzed by LC-MS/MS method.（3）Absolute quantitative amino acid metabolomics:cell samples from adenovirus-infected AGS 48h were collected and counted,divided into NC and Cag A groups,each with 5 biological replicates.Absolute quantitative metabolomics analysis targeting 20 amino acids was performed using gas chromatography–mass spectrometry（GC-MS）techniques.（5）Effect of overexpressing Cag A on DNA damage of gastric cancer cells.IF was used to detect the expression of Histone H2AX phosphorylation（γH2AX）.Flow cytometry was used to detect the cell cycle distribution and apoptosis of gastric cancer cells SGC-7901,AGS,WZK,and YHD that overexpress Cag A.Results:（1）The cag A recombinant adenovirus vector was successfully constructed.After the packaged p Adeno-cag A adenovirus infected primary gastric cancer cells,the cell morphology changed significantly in a“hummingbird-like”phonotype.（2）（1）WB results showed that compared with NC group,the expression of glycolytic metabolic enzymes HK2,ENO1 and GLUT1 in WZK and YHD cells overexpressing Cag A were down-regulated;PKM1/2 expression was up-regulated and LDHA expression was down-regulated in YHD cells.The differences were statistically significant（P<0.05）.（2）The stablely transfected cell lines AGS/glut1 and BGC-823/glut1 that overexpress glut1 were successfully established.IF results indicated that Cag A was expressed in the cell membrane and cytoplasm,co-localized with Glut1 on the cell membrane,resulting in reduced expression of Glut1 and formation of debris.（3）（1）The levels of ATP,glucose consumption and glucose uptake in AGS,YHD cells infected with p Adeno-cag A adenovirus for 48 h were significantly increased（P<0.05）,and ATP levels in BGC-823/glut1 and AGS/glut1 cells were also significantly increased（P<0.05）（2）The ATP rate results showed that,compared with the NC group,the rate of glycolytic ATP production increased in the cells overexpressing the Cag A group,and the rate of mitochondrial ATP production decreased in the primary cells YHD（P<0.05）.The mitochondrial function test showed that the mitochondrial basal respiration,Spare respiratory capacity and ATP production were significantly decreased in YHD cells overexpressing Cag A（P<0.0001）,and mitochondrial ATP production in AGS cells was reduced（P<0.05）.（4）Targeted energy metabolomics and metabolic flux analysis results showed that most of the tricarboxylic acid cycle metabolites were significantly downgraded,and some metabolites levels in glycolysis were up-regulated or unchanged;in amino acid absolute quantitative metabolomics,20amino acids were significantly up-regulated.（5）IF showed that the expression ofγH2AX in AGS and YHD cells infected with p Adeno-cag A was enhanced.Compared with the NC group,the percentage of cells in G2/M phase and the percentage of apoptotic cells of SGC-7901,AGS,YHD,and WZK cells increased significantly（P<0.05）.Conclusion:（1）Overexpression of Cag A downregulates the expression of glycolytic metabolic enzymes HK2,ENO1 and GLUT1 in gastric cancer cells.（2）H.pylori-Cag A mainly targets mitochondria,causing energy metabolism obstacles in mitochondria,reducing the reserve capacity of mitochondria,reducing the production capacity of aerobic oxidation,increasing glycolytic metabolism and amino acid metabolism,and inducing cell metabolic remodeling.（3）Cag A is genotoxic and can induce double-strand DNA breaks and promote apoptosis in cells,which may be related to gene instability in gastric cancer tissues.