| Helicobacter pylori(H.pylori)is a gram-negative rod-shaped bacterium that can colonize in gastric mucosa for a long time.It can cause gastric inflammation,atrophy,dysplasia and even cancer in human body.A variety of virulence factors produced by H.pylori may play a role in the bacterial pathogenicity.Among which cytotoxin-related gene A protein,namely Cag A protein,is a major virulence factor of H.pylori in the pathogenic process of infection.M2 isoform of pyruvate kinase(PKM2)is one of the key enzymes involved in glycolysis and gluconeogenesis.In the process of glycolysis,PKM2 catalyzes the synthesis of pyruvate from phosphoenolpyruvate,and then pyruvate enters The tricarboxylic acid cycle produces lactic acid to form ATP.This biochemical reaction is an important regulatory step in the glycolytic pathway.Current studies have found that PKM2 was up-regulated in various cancer tissues,such as cervical cancer,lung cancer and prostate cancer.At the same time,some studies have found that PKM2 was also highly expressed in gastric cancer tissues,which had an important influence on the development of cancer.However,in the process of H.pylori infection,it is still unclear whether Cag A protein promotes gastric mucosal carcinogenesis by affecting the expression of PKM2.And it is of great significance to explore this question to reveal the carcinogenic mechanism of H.pylori infection.ObjectiveIn this study,H.pylori NCTC11637 standard strain(Hp11637 cag A~+)and H.pylori NCTC11637 cag A gene knockout strain(Hp11637 cag A~-)were co-cultured with gastric epithelial cells,cag A gene transfected cells were tested,and an animal model of H.pylori infection in Mongolian gerbils was established,with aim to explore the regulatory effect of H.pylori and its cag A protein on the expression of PKM2 in gastric epithelial cells,and establish a basis for the study of the pathogenic mechanism of H.pylori infection.Methods1.H.pylori was co-cultured with cells:(1)The Hp11637 cag A~+strain was co-cultured with gastric mucosal epithelial GES-1 cells and gastric cancer SGC-7901 cells for 24 hours,and the multiplicity of infection(MOI)was 200:1,100:1 and 50:1,the expression level of PKM2 m RNA in the cells was detected.(2)The Hp11637 cag A~+and Hp11637 cag A~-strains were co-cultured with gastric mucosal epithelial GES-1cells and gastric cancer SGC-7901 cells at MOI=200:1 for 6h,12h,and 24h to detect the expression level of PKM2 m RNA in the cells.2.Plasmid transfection experiment:GES-1 cells and SGC-7901 cells were transfected with recombinant plasmid pc DNA-cag A and pc DNA 3.1(+)respectively.Cell samples were collected after transfection for 48h.The expression of Cag A protein in the transfected cells was detected by western blot and the expression level of PKM2m RNA was detected by q PCR.3.Animal experiment:60 SPF Mongolian gerbils were randomly divided into three groups(Hp11637 cag A~+group,Hp11637 cag A~-group and control group).The first two groups were given Hp11637 cag A~+and Hp11637 cag A~-by gavaging,and the last group were gavaged with sterile seed preservation solution,once every other day,for a total of five times.At 4,8,12 and 16 weeks after the last gavage,5 gerbils in each group were sacrificed.The stomach tissues of gerbils were taken,and the colonization of H.pylori in the stomach of gerbils was detected by urease test,H.pylori 16S specific gene detection and bacterial culture method.The m RNA expression level of PKM2 in the gastric epithelial cells of gerbils was detected by real-time fluorescence quantitative PCR.Results1.H.pylori was co-cultured with cells:(1)The Hp11637 cag A~+strain was co-cultured with GES-1 cells for 24 hours.The results showed that when the MOI of infection was 50:1,the expression level of PKM2 m RNA in the experimental group was not significantly different from that in the control group,when the MOI was 100:1and 200:1,the PKM2 m RNA level in the experimental group was significantly upregulated compared to the control group(P<0.001);when Hp11637 cag A~+strain was co-cultured with SGC-7901 cells,the expression of PKM2 in three different MOI groups was higher than that in the control group(P<0.001).(2)When Hp11637cag A~+and cag A~-strains were co-cultured with GES-1 cells at MOI=200:1 for 12h and24h,the expression levels of PKM2 m RNA of the cag A~-group and cag A~+group were significantly upregulated compared with the control group(P<0.01).When gastric cancer cell SGC-7901 was co-cultured with H.pylori at MOI=200:1 for 6 hours,12h and 24h,the expression level of PKM2 m RNA in the cag A~+group and cag A~-group were significantly increased compared with the control group(P<0.001).Moreover,compared with the cag A~-group,the expression level of PKM2 m RNA in the cag A~+group was also significantly up-regulated(P<0.01).2.Plasmid transfection experiment:Recombinant plasmid pc DNA-cag A and plasmid pc DNA 3.1(+)were used to transfect GES-1 and SGC-7901 cells,respectively.The corresponding cag A protein bands were successfully detected by Western blot after48h transfection,confirming that pc DNA-cag A was successfully transfected into the cells.And the results of q PCR showed that the expression of PKM2 m RNA in the pc DNA-cag A group was significantly higher than that in the pc DNA 3.1(+)group(P<0.001).3.Animal experiment:(1)H.pylori was detected in the stomach of both cag A~+and cag A~-groups of gerbils,while not detected in the control group;(2)After 4 weeks of infection of gerbils with Hp11637 cag A~+and cag A~-strains,the expression levels of PKM2 m RNA in the cag A~+and cag A~-groups were not significantly different from those in the control group(P=0.098,P=0.073,respectively);(3)After 8 weeks of infection of gerbils with Hp11637 cag A~+and cag A~-strains,the PKM2 m RNA expression levels in the two groups were significant higher than those in the control group(P<0.01);(4)After 12 weeks of infection of gerbils with Hp11637 cag A~+and cag A~-strains,the PKM2 m RNA expression level in the cag A~+group was significant higher than that in the control group(P<0.01),while the PKM2 m RNA expression level in the cag A~-group was not statistically different from the control group(P=0.0767),and the PKM2 m RNA expression level in cag A~+group was up-regulated when compared with cag A~-group(P<0.01);(5)After 16 weeks of infection of gerbils with Hp11637 cag A~+and cag A~-strains,the levels of PKM2 m RNA expression in the cag A~+and cag A~-groups were significant higher than those in the control group(P<0.01,P=0.014,respectively),and the PKM2 m RNA expression level in the cag A~+group was up-regulated when compared with the cag A~-group(P=0.0134).Conclusion1.Study of H.pylori co-cultured with cells and cell transfection experiments suggested that H.pylori Cag A protein could up-regulate the expression of PKM2 in normal gastric mucosa epithelial cells and gastric cancer cells.2.The animal model of H.pylori infection proved that the Cag A protein could up-regulate the expression of PKM2 in the course of chronic persistent with H.pylori infection.3.Previous studies have shown PKM2 was closely related to a variety of cancers,the results of this study suggested that Cag A protein might be involved in the occurrence and progression of gastric cancer by affecting the expression of PKM2 in gastric epithelial cells. |
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