| Helicobacter pylori(H.pylori)is a kind of gram-negative bacteria,which can colonize in the stomach for a long time,and related to gastritis,gastric and duodenal ulcer,gastric cancer and other gastrointestinal diseases.Hexokinase-2(HK2)is a key enzyme in glycolysis,catalyzing the conversion of glucose to glucose-6-phosphate,which is involved in cancer cell growth and metabolism.HK2 is overexpressed in colorectal cancer,liver cancer,lung cancer and other cancers,and may promote tumor progression through invasion,proliferation and migration of cancer cells.It has been reported that the expression level of HK2 is increased in gastric cancer,and it is related to the prognosis of gastric cancer patients,which may play an important role in the occurrence and development of gastric cancer.Studies have shown H.pylori CagA protein is closely related to the occurrence and development of gastric cancer.At present,it is not clear CagA protein is involved in the pathogenesis of gastric cancer by affecting the expression of HK2 in gastric epithelial cells.It is of great significance to explore the regulatory effects of H.pylori and CagA protein on the expression of HK2in gastric epithelial cells for the carcinogenic mechanism of H.pylori infection.ObjectiveIn this study,the co-culture experiment of H.pylori and gastric mucosal epithelial cells,the cell transfection experiment and the construction of the animal model of H.pylori infection were applied to explore the regulation effect of H.pylori and CagA protein on the expression of HK2 in stomach epithelial cells.This study can provide an important basis for the carcinogenic mechanism of H.pylori infection and the early diagnosis study of stomach cancer.Methods1.Identification of H.pylori NCTC11637 cag A deletion strain(H.pylori cag A~-):H.pylori NCTC11637 cag A~+and H.pylori cag A~-were cultured.They were extracted that the genomic DNA and CagA protein of the H.pylori NCTC11637 cag A~+and H.pylori cag A~-strains,and H.pylori NCTC11637 cag A~+and H.pylori cag A~-were identified by PCR and Western blot analysis.2.Co-culture experiment between H.pylori and gastric mucosal epithelium cells:With the MOI(multiplicity of infection)of 50:1,100:1 and 200:1,H.pylori 11637cag A~+strain was co-cultured with GES-1 and SGC-7901 cells for 24h,and the expression levels of HK2 m RNA were detected in cells.H.pylori cag A~+and H.pylori cag A~-strains were co-cultured with GES-1 and SGC-7901 cells for 6h,12h and 24h at the MOI of 200:1,and the expression levels of HK2 m RNA were tested by q PCR in cells.3.H.pylori cag A gene transfection cell experiment:The GES-1 and SGC-7901cells were transfected with plasmid pc DNA-cag A as the experimental group and plasmid pc DNA 3.1(+)was used to transfect GES-1 and SGC-7901 cells as the control group.The expression of HK2 m RNA was detected by q PCR and the expression of CagA protein was texted by Western blot for 48h.4.Mongolian gerbils experiment of H.pylori infection:Mongolian gerbils were randomly divided into three groups(H.pylori cag A~+group,H.pylori cag A~-group and control group).H.pylori cag A~+and H.pylori cag A~-group were gavaged with H.pylori NCTC11637 cag A~+and NCTC11637 cag A~-strain,respectively.The control group was gavaged with seed preservation solution,and attacked for five times with intervals of1 day.After 4,8,12 and 16 weeks of the last challenge,the gastric tissues of Mongolian gerbils were separated,and the HK2 m RNA expression level was detected by q PCR in the gastric mucosal epithelium cell of Mongolian gerbils.Results1.The identification of H.pylori NCTC11637 cag A deleted strain:H.pylori cag A~-strain does not contain the cag A gene and H.pylori NCTC11637 strain contain the cag A gene,which was identified by PCR and Western blot analysis.2.Co-culture experiment between H.pylori and gastric mucosal epithelium cells:(1)H.pylori cag A~+strain was co-cultured with GES-1 and SGC-7901 gastric mucosal epithelium cells for 24h,and the expression levels of HK2 m RNA were higher in the experimental groups than that in the control group(P<0.001)when MOI was50:1,100:1 or 200:1.The HK2 m RNA level at MOI=200:1 group was significantly increased compared with MOI=100:1 group and MOI=50:1 group(P<0.001).HK2m RNA levels of the MOI=100:1 group was significantly increased compared with the MOI=50:1 group(P<0.001).(2)H.pylori cag A~+ and cag A~-strain was co-cultured with GES-1 and SGC-7901cells for different hours:When H.pylori cag A~+ and cag A~-strains were co-cultured with GES-1 cell for 6h at the MOI of 200:1,the expression level of HK2 m RNA in the H.pylori cag A~+group was significantly increased compared with the control group(P<0.001);The expression levels of HK2 m RNA in the H.pylori cag A~+and control groups were higher than that in the H.pylori cag A~-group(P<0.001);When H.pylori cag A~+and cag A~-strains were co-cultured with SGC-7901 cell for 6h at the MOI of 200:1,the expression levels of HK2 m RNA in the H.pylori cag A~+and cag A~-group were significantly increased compared with the control group(P<0.001);Compared with the H.pylori cag A~-group,the expression level of HK2 m RNA in the H.pylori cag A~+group was no statistical difference(P>0.05).When H.pylori cag A~+and H.pylori cag A~-strains were co-cultured with GES-1 and SGC-7901 cells for 12h and 24h,the expression levels of HK2 m RNA of H.pylori cag A~+group and H.pylori cag A~-group were significantly increased compared with the control group(P<0.001),and in H.pylori cag A~+group was enhanced compared with H.pylori cag A~-group(P<0.001).3.Recombinant plasmid pc DNA-cag A transfection cell experiment:the GES-1and SGC-7901 cells were transfected with plasmid pc DNA-cag A and pc DNA 3.1(+),respectively.The CagA protein was found in gastric mucosal epithelium cells after transfection with plasmid pc DNA-cag A for 48 hours by Western blot.The expression level of HK2 m RNA in the pc DNA-cag A group was higher than that in the pc DNA 3.1(+)group(P<0.001).4.Detection results of stomach tissue specimen of H.pylori infection Mongolian gerbils:The HK2 expression levels were no significant difference in the H.pylori cag A~+and cag A~-group compared with the control group when the H.pylori infected Mongolian gerbils for 4 weeks(P>0.05),and in H.pylori cag A~+group was no statistical difference compared with H.pylori cag A~-group(P>0.05).H.pylori infected Mongolian gerbils for 8 weeks,and the results showed that the HK2 m RNA levels of the H.pylori cag A~+and cag A~-groups were increased compared with the control group(P<0.05),but in H.pylori cag A~+group was no significant difference compared with H.pylori cag A~-group(P>0.05).The HK2 expression level was higher in the H.pylori cag A~+group than that in the control group when the H.pylori infected Mongolian gerbils for 12 weeks(P<0.05),however,in H.pylori cag A~+and control groups were no significant difference compared with H.pylori cag A~-group(P>0.05).The HK2m RNA levels of the H.pylori cag A~+and cag A~-groups were significantly increased compared with the control group when H.pylori infected Mongolian gerbils for 16weeks(P<0.001).Moreover,the HK2 m RNA level in the H.pylori cag A~+group was significantly higher than that in H.pylori cag A~-group(P<0.001).Conclusion1.The results of H.pylori co-culture experiment and cell transfection experiment showed that H.pylori CagA protein could up-regulate the expression of HK2 in gastric mucosal epithelial cells.2.It was proved that H.pylori CagA protein could up-regulate the expression of HK2 in gastric epithelial cells in animal experiment.3.The results of this study suggested that H.pylori CagA protein may be involved in the development of stomach cancer by effecting the expression of HK2 in gastric mucosal epithelial cells.In addition,there may be other factors regulating the expression of HK2 in H.pylori in addition to CagA protein. |
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