Research On Proteomic And Effect Of Apoptin On Gastric Cancer Cell Lines SGC7901 Cell

Objective To construct eukaryotic expression vector pEGFP - N1 - VP3 and to stable transfect human gastric cancer cell line SGC-7901, To observe the distribution and subcellular 1ocation of fusion protein EGFP-VP3 in tumor cells,and to study the mechanism of Apoptin-induced apoptosis of tumor cells. The differentially expressed proteins between normal and VP3-induced apoptosis SGC-7901 cells were studied by two dimensional gel electrophoresis(2-DE)combining with mass spectral analysis.In the present study,we have applied the proteomics approach to study the effects of VP3 on the human gastric cancer cell line SGC-7901.Methods The VP3 gene fragment was amplified by PCR, and inserted into expression vector pEGFP-N1. After digested , the recombinant plasmid was then transferred into SGC-7901 cells by liposome. The distribution and subcellular 1ocation of fusion protein EGFP-VP3 in tumor cells were observed under fluorescence microscopy and laser scanning confocal microscope. The VP3 induced apoptosis of tumor cells in vitro was tested by AO/EB assay. Total proteins of VP3 treated cells and untreated cells were extracted and separated by two dimensional gel electrophoresis (2-DE) . Conditions(gel strip : pH3-10 / pH4-7 ; SDS-PAGE concentration:10%/12%;electrophoresis time:6h/7h)were adjusted,the differential expression proteins were analyzed with PDQuest software,and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS)and Mascot database searching.The mRNA alterations of ICEBERG were confirmed by semi-quantitative RT-PCR,ICEBERG proteins were confirmed by Western blot.Results Enzyme digestion analysis and DNA sequencing showed that the target gene was cloned into recombinant vector. High expression of EGFP-VP3 was detected in the transfected tumor cells, and EGFP-VP3 moved from cytoplasm to nucleus gradually ,finally accumulated in nucleus.Apoptosis of tumor cells induced was observed after transfection by AO/EB assay. 2-DE patterns of SGC-7901 cells with high-resolution and reproducibility were obtained.The results indicated that there were 862±36 and 648±27 protein spots detected in the 2-DE maps from normal and VP3 transfected SGC-7901 cells,respectively.The distribution of the protein spots inthe two maps was similar.A total of 8 differentially expressed proteins were visualized by 2-DE and silver stain.Of these,6 proteins were identified via MALDI-TOF-MS analysis. At the mRNA and protein level,Human ATP synthase subunits, mitochondrial (ATP5S),Caspase-1 inhibitor Iceberg (ICBR),Trafficking protein particle complex subunit 6A( TPC6A),Transaldolase( TALDO),Barrier-to-autointegration factor (BAF),Retinol dehydrogenase 13(RDH13) were downregulated alter treated by VP3.Conclusion Constructing the eukaryotic expression vector pEGFP-N1-VP3 successfully, and The positive SGC-7901 cell clones expressing the large number of green fluorescent protein and fusion protein EGFP-VP3 stably were obtained.The fusion protein EGFP-VP3 has nucleic 1oealization function in tumor cells and can induce the apoptosis of tumor cell. Several proteins expressed differentially in SGC-7901 cells after treated by VP3,which take part in some signal pathways of cell proliferation,differentiation,apoptosis,etc,the antitumor effect of VP3 may relate to the interplay of the above proteins.