Regulation Of H.pylori CagA Protein On The Expression Of Serine/threonine Kinase PIM2 In Gastric Cancer Cells

ObjectiveThis work is designed to demonstrate the effect of H.pylori CagA protein on the expression of serine/threonine kinase PIM2 in gastric cancer epithelial cells by constructing a H.pylori strain with cagA gene knocked out,co-culturing cells with H.pylori strains,transfecting cells with cagA gene recombinant plasmid,with aim to lay basis for approaching pathogenesis of H.pylori infection as well as diagnosis and treatment of gastric cancer.Methods1.The construction of H.pylori without cagA gene:(1)The upstream and downstream homologous fragments of cagA gene from H.pylori NCTC11637 and chloramphenicol resistance gene from the plasmid pNZ8110 were amplified by PCR,then all the fragments were inserted into plasmid pBluescript II SK(-)by the seamless cloning technology to construct the isogenic cagA gene knock-out mutant vector,PCR,enzyme digestion and sequencing were used to identify the recombinant plasmid.(2)The mutant vector was transformed into NCTC 11637 by electroporation,and the mutant strain were selected on chloramphenicol agar plate.Using PCR,sequencing and Western blot to identify the deletion strain NCTC 11637?cagA.2.H.pylori was co-cultured with gastric cancer cells:(1)With the MOI of 50:1,100:1 and 150:1,gastric cancer cells HGC27,SGC7901 or AGS were co-cultured with H.pylori NCTC 11637,assaying the expression of PIM2 mRNA in cells co-cultured for 24h.(2)With MOI of 150:1,HGC27,SGC7901 or AGS cells were co-cultured with NCTC11637 for 6h,12h and 24h,respectively,then the expression of PIM2 mRNA was detected by qPCR.(3)Gastric cancer cells HGC27,SGC7901 or AGS co-cultured with NCTC11637 or NCTC11637?cagA for 24h at the MOI of 150:1,then the expression of PIM2 mRNA was tested.3.Construction of the eukaryotic expression vector of H.pylori cagA gene and cellular transfection:(1)Using PCR technique to gain the cagA gene fragment from the genomic DNA of NCTC11637,the plasmid pcDNA3.1(+)and seamless cloning technique were utilized obtain the eukaryotic expression vector pcDNA-cagA.(2)HGC27,SGC7901 or AGS cells were transfected with pcDNA-cagA after the recombinant plasmid was identified successfully,with the empty vector pcDNA3.1(+)as the control.(3)The expression of PIM2 mRNA was tested by qPCR when cells transfected for 48h.Results1.The identification of H.pylori cagA gene mutant strain:After the identification by PCR and Wentern blot,H.pylori with cagA gene knocked out had been constructed successfully2.The results of the expression of PIM2 in gastric cancer cells co-cultured with H.pylori:(1)With the different values of MOI,co-culturing H.pylori with gastric cancer cells for 24h:?The PIM2 expression levels of HGC27 cells co-cultured with NCTC11637 were significantly increased when the MOI was 100:1(P=0.001)or 150:1(P<0.001)compared with the control group;?As for SGC790 cells co-cultured with NCTC11637,the expressions of PIM2 mRNA in the four experimental groups were all higher than that in the control(P<0.001);?When AGS cells co-cultured with NCTC11637S the expression of PIM2 was increased when MOI was 150:1(P<0.001).(2)With the MOI of 150:1,co-culturing H.pylori with gastric cancer cells for different hours:?The expression of PIM2 mRNA in HGC27 cells co-cultured with NCTC11637 was up-regulated at 12 h and 24 h(P<0.01);?The expression of PIM2 in SGC7901 cells co-cultured with NCTC11637 was up-regulated at 6h,12h and 24h(P<0.05);?The expression of PIM2 mRNA in AGS cells co-cultured with NCTC11637 for 6h,12h or 24h was higher than that in the control group(P<0.001).(3)NCTC11637 and NCTC11637?cagA were co-cultured with HGC27?SGC7901 or AGS cells at MOI of 150:1 for 24h:The expressions of PIM2 mRNA in the defferent gastric cancer cells co-cultured with NCTC11637 or NCTC11637?cagA were higher than that in the control(P<0.01),and the expression of PIM2 in cells co-cultured with NCTC11637 was higher than that co-cultured with NCTC11637 ?cagA(P<0.001).3.The eukaryotic expression vector construction of NCTC11637 cagA gene and cell transfection:(1)The eukaryotic expression vector pcDNA-cagA was successfully constructed,which was conformed by PCR and sequencing;(2)HGC27,SGC7901 and AGS cells were transfected by pcDNA-cagA,and the expression of cagA gene was detected by qPCR and Western blot,which confirmed the successful transfection of pcDNA-cagA into cells;(3)The expression levels of PIM2 mRNA in HGC27,SGC7901 and AGS cells,which were transfected for 48 hours,were significantly higher than that in the control group(P<0.05).Conclusions1.H.pylori with or without cagA gene can up-regulate the expression of PIM2 in gastric cancer cells significantly;And the expression level is related to the MOI and duration of infection.2.H.pylori cagA gene can enhance the regulation of H.pylori on the expression of PIM2 in gastric cancer epithelial cells.3.The cagA gene transferred into gastric cancer cells can up-regulate the expression of PIM2 in cells,further confirming the regulatory effect of H.pylori cagA gene and its expression protein on the expression of PIM2 in gastric cancer epithelial cells.In view of the fact that PIM2 is a proto-oncogene,the results suggest that cagA gene may play an important role in the pathogenesis of H.pylori infection.