Verificate And Analyse The Differentially Expressed Genes In Over-expressed E2F-1/Cdx2 Gastric Cancer Cell DNA Microarray By Semi-quantitive RT-PCR

Gastric Carcinoma is one of the frequent humanity's malignant tumours. All over the world, The incidence rate of gastric carcinoma is the third among man's manlignant tumors, and the mortality is after the pulmonary carcinoma at the second. Most patients have been progressing to advanced stage or have distant aversion or have clinical symptoms at the time of diagnosis. The rate of radical resection is very low. At present, Having no normative and effective treatment perscription postoperation, such as radiotherapy chemotherapy have little effects, even exert great toxico-side effect to patients, and the survival rate after five year is lower. Therefore the neo-promising therapy has been badly requested. Recently, many of researchs have been done on malignant tumours and the study of cell cycle have derived breakthroughly advancement. The transcription factor E2F-1 is capable of regulate mechanisms of Rb/E2F path and cell cycle inducing apoptosis in many cells, and has the potential to function of tumour suppressor; Cdx2 protein contains 311 monamino acid, through helico-loop-helico combine to the corresponding region in the form of transcription factors to regulate the expression of DNA. Lucubrated of E2F-1/Cdx2 in the role of gastric cancer cells as well as with other tumor-associated genetic changes and the mechanism of interaction. Can not only clarify the mechanism of E2F-1/Cdx2 effect on gastric cancer, but also assist in the diagnosis and classification of gastric cancer, and provide a reliable theory for treatment. Objective:Verify and analyse of differential expression gene in DNA microarray by RT-PCR,in order to further explore the impact of over-expressed E2F-1/Cdx2 on the biological behavior mechanism of gastric carcinoma cell.Method1.liposome Lipofectamine 2000 transfected pCMV-E2F-1-HA2, pCMV-Cdx2-A2,pCMV-HA2 eukaryotic expression vector to the MGC-803 gastric carcinoma cells, then extract Total RNA and protein from transfected cells in each group. Using RT-PCR and Western Blot techniques to Verify pCMV-E2F-1-HA2, pCMV-Cdx2-HA2, pCMV-HA2 plasmids have transfected into cells;2.Extracted and purified total RNA of MGC-803/E2F-1, MGC-803/Cdx2, MGC-803/HA and MGC-803 gastric carcinoma cell. Using reverse- transcriptase method to synthesis cDNA, combined with PCR reaction to amplify the differential expression gene fragment. Used semi-quantitative the method of Gel imaging system gray-scale ratio to obtain the data of RT-PCR;3. Through analysised the data of RT-PCR in each group, Gained the result of RT-PCR compared with the differential expression gene in DNAmicroarray.Results1. Have detected the mRNA and fusion protein expressed in two groups of MGC-803/E2F-1,MGC-803/Cdx2 gastric carcinoma cell;2. The result of RT-PCR is consistented with the differentially expressed genes in DNA microarray.Conclusion1. Have been successfully transfected with the overexpression of E2F-1/Cdx2 gene into gastric carcinoma MGC-803 cells line;2. The results of MGC-803/E2F-1 RT-PCR is consistented with the five differential expressed genes in over-expressed E2F-1/Cdx2 gastric carcinoma cell DNA microarray;3. The results of MGC-803/Cdx2 RT-PCR is consistented with the six differential expressed genes in over-expressed Cdx2 gastric carcinoma cell DNA microarray;4. Overexpression of E2F-1/Cdx2 gene caused different gene expression in gastric carcinoma MGC-803 cells line. These differentially expressed genes through various biological signaling pathways involved in gastric carcinoma cell proliferation, growth, apoptosis and the regulation of invasion and metastasis. Subsequently, con-effect on the change of gastric carcinoma cell biological behavior.