Construction Of DEC1Eukaryotic Over-expression Plasmid And Its Effect On The Proliferation Of Gastric Cancer Cells

ObjectiveGastric cancer is one of the most frequent malignant tumor in the world, its incidence and mortality rates in the world and in China both the second. The origin and development of gastric cancer of molecular mechanism in life science research has been a hot issue.Transcription factors anomalies may participate in the occurrence of its development.Differentiated embryo-chondrocyte expressed gene1is a mumber of bHLH protein family and has a basic helix-loop-helix structure domain.DEC1has been shown to be involved in many normal tissue and closely related with the occurrence of normal circadian rhythms, autoimmune and the stability of the Immune environment.DEC1promoter area including kinds of GC-box,5’end including multiple transcription starting sites,including cAMP elements and many kinds of GC-box. DEC1widely present in the most normal tissue,and articipate in cell proliferation, differentiation, tumors and immune adjustment. Growth factor, UV, nutrition, low oxygen, infection and hormone can influence the expression of DEC1gene. There are high expression of DEC1in many tumor cells such as leukemia, colon, lung adenocarcinoma, glioma, kidney DEC1protein is composed of412amino acids, Widely present in the most normal tissue, and through the combined with target gene promoter of E-box sequence or through the protein-protein of interaction play transcriptional regulation activity. Gastric cancer MGC-803cells are the objects, pGEM as a carrier and construst the recombinant pcDNA3-DEC1, pcDNA3-DEC1was transfected into MGC-803cell, observe the DEC1gene expression after transfection.So we can detect the cignificance of over expression DEC1. Furthermore,we can investigate the relationship of DEC1and gastric cancer.Methods1.The human gastric cancer cell line MGC-803were cultured and passed;2. DEC1gene cDNA was amplified from gastric cancer MGC-803cell by RT-PCR3. Construst recombinant pcDNA3-DEC1Eukaryotic expression vector and pcDNA3-DEC1was transfected into MGC-803cell. MGC-803cells in the exponential phase of growth were divided into three groups(pcDNA3-DEC1group, negative control group, blank control group);4. RT-PCR detected DEC1mRNA expression inhibited in three groups;5. Western blot detected DEC1expression inhibited in three group;6. The proliferation was analyzed by MTT and Immunocytochemistry in three group.Result1. pcDNA3-DEC1was successfully constructed;2.DEC1mRNA expression in MGC-803cells of pcDNA3-DEC1group was increased than negative control group and blank control group(p<0.05);3.DEC1protein expression in MGC-803cells of pcDNA3group was increased than negative control group and blank control group(p<0.05);4. MTT assay and Immunocytochemistry indicated that proliferation was increased in pcDNA3-DEC1group.Conclusion 1. The pcDNA3-DEC1plasmid was identified with the method of PCR and sequencing;2. As a result of pcDNA3-DEC1was transfected into MGC-803cell,DEC1mRNA and protein expression was increased;3. Expression of DEC1gene can promote cell proliferation of gastric cancer cell MGC-803.