The Effects Of Cdx2 And E2F-1 On Biological Characters Of Human Gastric Carcinoma Cell Line

The incidence rate of gastric carcinoma is also in the first position among manlignant tumors in our country,and the mortality is about one quarter of global ones.Most patients have been progressing to advanced stage at the time of diagnosis.The rate of radical resection is lower,treatments including radiation, chemotherapy or preoperative chemotherapy have little infuence,even exert great side effect to patients.And at present having no normative chemotherapy, therefore the gene therapy has been introduced and displayed its promising.Two important properties of cancers are able to deregulate cell proliferation and suppress cell death.Cdx2 is a specific nuclear transcriptional factor,First isolated by Mlodzik and co-workers in Drosophila gene caudal.This gene encodes a transcriptional factor that plays an important role in the development and homeostasis of the intestinal epithelium.In the present study,Cdx2 expression can inhabit the invasiveness and metastasis of colon carcinoma. E2F-1 is the prototype of the E2F family of transcription factors.Thus far,eight members(E2F-1 to E2F-8)of the E2F family of DNA-binding proteins have been identified.E2F-1 play a crucial role in the cell cycle.Initially,E2F-1 was suggested to behave as an oncogene because it promotes cellular proliferation by stimulating the expression of a number of genes that promote the transition from G1 to S phase.And E2F-1 overexpression stimulates quiescent cells to enter into S phase.In addition,the overexpression of E2F-1 in HNSCC cell lines can stimulate cell cycle re-entry but is also associated with increased invasiveness,suggesting a positive role of E2F-1 in cell cycle progression as well as tumor progression and metastasis.On the other hand,the overexpression of E2F-1 has also been shown to induce apoptosis in several cell types,indicating that in addition to its critical role in regulating cell growth,E2F-1 also participates in coordinating programmed cell death.Recent studies with E2F-1 knockout mice have suggested that E2F-1 functions as a tumor suppressor1.Method1.1 Transient transfection and expression of gastric tumor cellsGastric carcinoma MGC-803 cells were transfected with Lipofectamine 2000.Total RNA and protein of cells were extracted respectively at the time of 48h after tranfected,methods of RT-PCR and Western blot were used to identify the expression of E2F-1 and Cdx2.1.2 The assays after transient transfection in vitroThe assays were divided into three groups,The MGC-803 cells transfected by pCMV-Cdx2-HA and pCMV-E2F-1-HA were experimental groups,the cells transfected by pCMV-HA was blank control and MGC-803 cells untransfected was negative control.Cell matrigel invasion assay,adhesion assay,scratching assay,cloning assay,MTT method and apoptosis rate were used to study the effects of Cdx2 gene.Cell matrigel invasion and migration assays,adhesion assay,cloning assay were used to study the effects of E2F-1 gene.2.Results2.1 Transient expression of E2F-1 and Cdx2 after transfected.After transfeted 48h,mRNA and protein of E2F-1 and Cdx2 were detected with RT-PCR and Western blot.2.2 All assays showed that the invasiveness,migration,adhesion and proliferation of MGC-803 cells transfected by pCMV-Cdx2-HA were less than those of MGC-803 cells transfected by pCMV-HA and untransfected cells. Apoptosis rate of MGC-803 cells transfected by pCMV-Cdx2-HA was higher than that of MGC-803 cells transfected by pCMV-HA and untransfected cells.And the assays showed that the invasiveness,migration and proliferation of MGC-803 cells transfected by pCMV-E2F-1-HA were less than those of MGC-803 cells transfected by pCMV-HA and untransfected cells.However, E2F-1 did not affect the adhesive ability of MGC-803 cells with the matrix.3.ConclusionThe eukaryotic expression vector pCMV-E2F-1-HA and pCMV-Cdx2-HA have been successfully transfected,they can be expressed in the gastric carcinoma cells.All assays suggest that E2F-1 and Cdx2 can affect biological characters of human gastric carcinoma cell line MGC-803.They may depress the invasion and metastasis of gastric carcinoma cell.