Stable Transfection Of Sv40tag Gene And Its Biological Effects On Human Gastric Carcinoma Cells Sgc7901

SV40 is a kind of DNA tumor viruses,which can transform cells and induce tumorigenesis in animals in vitro.The presence and expression of SV40 gene were detected in many kinds of human tumors,indicating that SV40 are associated closely with human carcinogenesis.SV40 transforming activity and etiopathogenesis are mostly related to the expression of the early region of viral genome encoding for T antigen(Tag).SV40Tag can interact with tumor suppressors p53 and Rb inactivating their functions,the inactivation of p53 and Rb may be an important mechanism in the pathogenesis of human tumors.Tag can integrate into the cell genome and destroy the normal structure and stability.It may transactivate many cellular genes expression, stimulating DNA replication.SV40Tag plays an decisive role in the cell transformation,which related closely with carcinogenesis and cell proliferation. SV40Tag transgenic mice models could be induced many kinds of tumors,which suggested that SV40Tag gene transgenic mice can be used for the research of the exploration of tumorigenesis mechanisms.SV40Tag transgenic mice models has been produced by use of tissue specific expression promoters.H⁺/K⁺ATPase specifically expressed in gastric parietal cells. The animal model of gastric cancer carrying SV40Tag gene can be established using H⁺/K⁺ATPaseβsubunit promoter,and can be applied for the study on the pathogenesis of gastric cancer,providing a powerful tool to prevent and treat gastric cancer. Based on the previous study,Eukaryotic expression vectors pcDNA3.1(-)/HKSV which containing SV40Tag gene were transfected into SGC7901,EC9706 and KB cell lines.After transfection,the stable transfected cell lines were established after screening by G418 and expanded in culture.PCR,RT-PCR and Immunocytochemistry methods were used to detect the integration and expression of SV40Tag gene in stable transfected cell strains.The effects of SV40Tag gene expression on the biological characteristics of SGC7901 cells were observed.This study investigated the mechanism of SV40Tag and the relationships between SV40Tag gene expression and the occurrence,development of tumors,providing an experimental and theoretical basis for the establishment of transgenic mice models.Materials and methods:1.The plasmids enzyme digestion identification These plasmids pcDNA3.1(-),pcDNA3.1(-)/HKSV and pcDNA3.1(-)/SV were confirmed by restriction enzyme digestion analysis.These plasmids were digested with BamHâ… .2.Transfection Eukaryotic expression vectors which containing SV40Tag gene pcDNA3.1(-)/HKSV and pcDNA3.1(-)/SV were transfected into SGC7901,EC9706 and KB cell lines using Lipofectamine^TM2000.Positive colonies were screened by the selection of G418 and expanded in culture,obtaining stable transfected cell strains.3.Identification of SV40Tag gene stable transfected cell lines After stable transfection,the integration and expression of SV40Tag gene were studied at DNA,RNA and protein levels.PCR analysis was performed using SV40Tag gene specific primer for detection of SV40Tag gene after isolation of genome DNA from transfected and untransfected cells.The total RNA was extracted from cells, and SV40Tag gene mRNA expression was identified by RT-PCR. Immunocytochemistry staining was used to detect the expression of SV40Tag in stably transfected and untransfected cells. 4.The biological characteristics of SGC7901 transfected by SV40 Tag gene The proliferation activity in the transfected and nontransfected SGC7901 cells were identified with MTT assay.Flow cytometry was applied to investigate cell cycle.5.Statistical analysis SPSS10.0 statistical software was used to analyze experimental data.To numerical variable,the data were expressed with(?)±s,ANOVA was used to compare these data,α=0.05 was selected as test level.Results:1.The restriction enzyme digestion results pcDNA3.1(-)/HKSV was digested by BamHâ… into two DNA fragments(2.7kb and 6.4kb).pcDNA3.1(-)/SV was digested by BamHâ… into 2.7kb and 5.4kb two fragments,and pcDNA3.1(-) was digested by BamHâ… into 5.4kb DNA fragment.This results matched with theoretical values, providing reliable basis for further researches.2.PCR 443bp GAPDH DNA bands were detected in every cell group.618bp DNA fragments specific for SV40Tag gene were observed in pcDNA3.1(-)/HKSV and pcDNA3.1(-)/SV transfected SGC7901,EC9706 and KB cells,but not in pcDNA3.1(-) transfectant and untransfected cells.3.RT-PCR 443bp GAPDH DNA bands were found in every cell group.272bp fragments expected for SV40Tag gene were observed in pcDNA3.1(-)/HKSV and pcDNA3.1(-)/SV stable transfected SGC7901,EC9706 and KB cells,but not in pcDNA3.1(-) transfectant and nontransfected cells.4.Immunocytochemistry staining Positive expression of SV40Tag were present in pcDNA3.1(-)/HKSV and pcDNA3.1(-)/SV stable transfected SGC7901,EC9706 and KB cells,but not in pcDNA3.1(-) transfected and untransfected cells.5.MTT assay SV40Tag gene transfected SGC7901 cells grew much faster in vitro compared with the empty plasmids transfectant and untransfected cells(P＜0.05).6.FCM Detection Cellular proportion of G₁ phase in pcDNA3.1(-)/HKSV and pcDNA3.1(-)/SV transfected SGC7901 cells were lower than that of empty plasmid transfected and untransfected cells,while the cell proliferation index increased in pcDNA3.1(-)/HKSV and pcDNA3.1(-)/SV stable transfected SGC7901 cells(P＜0.05).Conclusions:1.SV40Tag gene expressed steadily in pcDNA3.1(-)/HKSV stable transfected SGC7901,EC9706 and KB cell lines.2.SV40Tag gene stable transfection can promote the proliferation of SGC7901 cells.