| ObjectiveIn this study,H.pylori Mel-Hp27 cagA deletion strain,bacteria and gastric cancer cells co-culture and cell transfection experiments were constructed to identify the regulatory effect of H.pylori and cagA on TPI expression in gastric cancer cells,and to further reveal the molecular mechanism of H.pylori infection promoting the development of gastric cancer.Methods1.Construction of H.pylori Mel-Hp27 cagA deletion strain(Hp27 cagA-):(1)Construction of cagA target vector:The genomic DNA of isolated strain Mel-Hp27(Hp27 cagA+)from zhengzhou was used as template to amplify the homologous arms Fupand Fdown on both sides of cagA.The chloramphenicol resistance gene fragment Fcm was amplified with pNZ8110 plasmid as template.Through the seamless cloning technology,the Fup,Fdown,Fcm and pBluescript ? SK(-)plasmid was built into targeting carrier pBlK-mutant-cagA;(2)Construction of cagA deletion strain:the target vector pBlK-mutant-cagA was transformed into the receptor Hp27 by electric transformation,and the cagA deletion strain was obtained by homologous recombination.(3)Identification of cagA deletion strain:the genomic DNA and bacterial proteins of the cagA gene deletion strain Hp27 cagA-and the original Hp27 cagA+ were extracted,and the knockout effect of cagA was identified by PCR and Western blot analysis.2.Co-culture experiment between bacteria and gastric cancer cells:(1)Hp27 cagA+strain was co-cultured with gastric cancer cells AGS and SGC-7901 for24h with the Multiplicity of infections(MOIs)of 50:1,100:1 and 200:1,respectively,to detect the expression level of TPI mRNA in gastric cancer cells.(2)Hp27 cagA+and Hp27 cagA-strains were co-cultured with AGS and SGC-7901 gastric cancer cells with MOI=200:1 for 6h,12h and 24h,respectively,to detect the expression level of TPI mRNA.3.Cell transfection experiment with recombinant plasmid pcDNA-cagA:recombinant plasmid pcDNA-cagA and plasmid pcDNA 3.1(+)were used to transfect AGS and SGC-7901 cells,respectively,and the expression level of TPI mRNA was detected by qPCR and the expression of CagA protein in the transfected cells was detected by Western blot after transfection for 72h.4.Construction and transfection experiment of gene site-directed mutation plasmids pcDNA-cagA-BM and pcDNA-cagA-DM:(1)Design mutation primers according to the sequence of EPIYA-B and EPIYA-D tyrosine phosphorylation sites of CagA proteins,and carry out site-directed mutation on the target plasmid pcDNA-cagA by using the mutation primers and Phanta Max high-fidelity DNA polymerase;Mutant plasmids pcDNA-cagA-BM and pcDA-cagyA-DM were extracted,and then DNA sequencing was performed on the plasmids.(2)Mutated plasmids pcDNA-cagA-DM,pcDNA-cagA-DM,recombinant plasmids pcDNA-cagA and plasmid pcDNA 3.1(+)were transfected into SGC-7901 cells,and the expression level of TPI mRNA was detected after transfection for 48h.Results1.PCR and Western blot identification results of Hp27 cagA deletion strain(Hp27 cagA-)showed that the Hp27 cagA had been knocked out and the Hp27 cagA deletion strain was successfully constructed.2.Co-culture experiment between bacteria and gastric cancer cells:(1)Hp27 cagA+strain was co-cultured with AGS and SGC-7901 gastric cancer cells for24h,and the results showed that when MOI=50:1,100:1 and 200:1,TPI mRNA levels of the experimental groups were significantly increased compared with the control group(P<0.001).Moreover,the TPI mRNA level at MOI=200:1 was significantly higher than that at MOI=100:1(P<0.001),the TPI mRNA level at MOI=100:1 was significantly higher than that at MOI=50:1(AGS:P<0.001;SGC-7901:P<0.01).(2)When Hp27 cagA+and cagA-strains were co-cultured with AGS cells with MOI=200:1 for 6h,the expression levels of TPI mRNA in the cagA+and cagA-groups were significantly up-regulated compared with the control group(P<0.01,P<0.001),the cagA+group significantly decreased compared with the cagA-group(P<0.01);When Hp27 cagA+and cagA-strains were co-cultured with AGS cells for 12h and 24h,the expression levels of TPI mRNA of the cagA-group and cagA+group were significantly up-regulated compared with the blank control group(P<0.001),and the expression level of TPI mRNA in cagA+group was up-regulated compared with that in cagA-group(P<0.001).When the bacteria and SGC-7901 cells were co-cultured with MOI=200:1 for 6h,TPI mRNA expression levels of cagA+group and cagA-group were higher than that of the blank control group(P<0.001),and TPI mRNA expression level of cagA +group higher than that of the cagA-group(P<0.01);When co-cultured for 12h and 24h,the expression levels of TPI mRNA in the cagA-and cagA+ groups were significantly up-regulated compared with the blank control group(P<0.001),TPI mRNA expression level was up-regulated in the cagA+ group compared with the cagA-group(P<0.001).3.Recombinant plasmid pcDNA-cagA transfection experiment:The recombinant plasmid pcDNA-cagA and plasmid pcDNA 3.1(+)were used to transfect AGS and SGC-7901 cells,respectively.The results of qPCR showed that the expression level of TPI mRNA in pcDNA-cagA group was significantly higher than that in pcDNA 3.1(+)group(P<0.001).4.Construction and transfection experiments of mutant plasmids pcDNA-cagA-BM and pcDNA-cagA-DM:(1)Sequencing of recombinant plasmids with site-directed mutations showed that site-directed mutations of EYIYA-B and EYIYA-D tyrosine phosphorylation sites of MEL-Hp27 CagA proteins were successful.(2)Mutated plasmid pcDNA-cagA-BM/DM,recombinant plasmid pcDNA-cagA and empty plasmid pcDNA 3.1(+)were transfected into SGC-7901 cells,respectively.After transfection for 48h,the detection results showed that the expression levels of TPI mRNA in the mutated plasmid pcDNA-cagA-BM and pcDNA-cagA-DM were significantly down-regulated compared with the pcDNA-cagA group(P<0.001),and not statistically different from that in the empty plasmid group(P=0.94,P=0.61).Conclusion1.H.pylori CagA has the effect of regulating TPI expression in gastric cancer cells.As TPI is closely related to the occurrence and development of various tumors,the findings of this study suggest that CagA may be involved in the development of gastric cancer by regulating the expression of TPI in gastric tissue cells.2.The effect of H.pylori CagA on TPI expression in gastric cancer cells is regulated by tyrosine phosphorylation.3.Co-culture time and MOI of H.pylori and gastric cancer cells affect the expression level of TPI in gastric cancer cells.It was observed for the first time that H.pylori cagA gene may reverse regulate the expression of TPI when the co-culture time between H.pylori and gastric cancer cells was different.4.The study found that cagA-strain infection up-regulated the expression of TPI in gastric cancer cells,suggesting that there were other factors regulating the expression of TPI in H.pylori besides the CagA protein. |
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