Effects Of DRAM Gene On Proliferation,Apoptosis And Autophagy Of Gastric Cancer SGC7901 Cells

Objective:①To construct the plasmid containing overexpressed damage-regulated autophagy modulator(DRAM) which is a downstream gene of p53 and produce reco-mbinant adenovirus Admax-pDC315-DRAM-EGFP.②Transfect gastric cancer SGC7901 cells with Admax-pDC315-DRAM-EGFP to observe its effect on cancer cell proliferation,apoptosis and autophagy.Methods:①Acquisition target gene DRAM from the cDNA library with PCR method. Both the target gene and eukaryotic expression vector pDC315-EGFP were digested with EcoR-I. The digestional products were electrophoresis directio-nal connected and transformed into bacterial competent cells. Sequence and comp-are the positive clones which were identified by PCR to the right purpose, so as to construct plasmids pDC315-DRAM-EGFP. Cotransfected the HEK293 cells withthe shuttle plasmids pDC315-DRAM-EGFP and packaging plasmids pBHGF35. After generated by homologous recombination, the recombinant adenovirus Admax-pDC315-DRAM-EGFP was generated through AdMax? system. Determine titer ofrecombinant adenovirus, which were amplified by 293 cells and purified. Choice the best multiplicity of infection(MOI), and calculate the transfectional efficiencyat this MOI.②Transfect human gastric cancer SGC7901 cells with Admax-pDC 315-DRAM-EGFP and observe morphologic changes of them under microscope. The MTT assay was used to determine the cytotoxic effect of Admax-pDC315- DRAM-EGFP. We applicated LC3 immunofluorescence to detect the autophagy- specific protein LC3 changes in expression and distribution in SGC7901 cells; andwe also used Western-blot to determin p53, Bcl-2, beclin-1 protein expression in gastric cancer SGC7901 cells after treated by Admax-pDC315-DRAM-EGFP.Results:①The products of digestion and directional connection were gene sequ-enced. The result was consistent with the purpose of sequences, which suggest that DRAM gene was correctly inserted into the eukaryotic expression vector pDC315-EGFP. Recombinant adenovirus Admax-pDC315-DRAM-EGFP containing DRAM which can transfect SGC7901 cells was successfully constructed by AdMax? system. Titer of purified recombinant adenovirus was 2.8×109 IU/ml. Efficiency of transfection (green fluorescent cells / total cells) up to the peak(93±5.4%) when the multiplicity of infection (MOI)equal to 60.②After Admax-pDC315-DRAM-EGFP transfect SGC7901 cells, the inhibitory effect on cellular proliferation became more and more obvious with time increased(after 40μl Admax-pDC315-DRAM-EGFP treated SGC7901 cells for 24h, 48h and 72h, the cellular proliferational inhibition rates were 12.69±2.43%, 38.49±1.31% and 69.15±2.50% respectively, which were significantly different (P<0.05) to negative control group. As time prolonging , cellular autophagy-specific protein LC3 expression increased and had diffuse trend in distribution; protein expression level in control group,negtive virus group,recombinant adenovirus 24h group and recombinantadenovirus 48h group determined by Western Blot and caculated with SigmaScan Pro 5 software were respectively p53: 1912416,2614012,3272305,4982792(rela-tive ratio was 1/1.37/1.71/2.61); beclin-1: 678110,593149,1149591,987640(rel-ative ratio was 1/0.87/1.70/1.46); bcl-2: 362689,346132,227493,160150(relative ratio was 1/0.95/0.63/0.44)。Conclusion : The recombinant adenovirus Admax-pDC315-DRAM-EGFP can succes-sfully transfect gastric cancer SGC7901 cells. After transfection, cancer cellular pr-oliferation of SGC7901 cells was obviously inhibited; expression of LC3 which is autophagy-specific protein gradually enhanced; beclin-1 which is the key proteinto active autophagy and p53 protein expression was gradually increased while bcl-2 protein which inhibits apoptosis obviously decreased. So we infered that DRAMgene can activated autophagy, up-regulate the level of p53-mediated apoptosis ingastric cancer SGC7901 cell line.