Construction of DRAM gene eukaryotic expression vector and effect of the expression on HepG_2 cells

Objective To construct damage-regulated autophagy modulator(DRAM)gene full-length cDNA eukaryotic expression vector pEGFP-DRAM and to assess effect of the expression on human HepG2 cells.Methods The full-length cDNA encoding DRAM gene was amplified by reverse transcription polymerase chain reaction(RT-PCR) and cloned to eukaryotic expression vector pEGFP-N1,the recombinant pEGFP-DRAM was identified by double digestion with restriction endonucleases and sequencing.The recombinant plasmid was transfected into HepG2 cells by lipofectamine method.The expression of DRAM mRNA in HepG2 cells was assayed by laser scanning confocal microscopy(LSCM).Cell apoptosis,cell cycle and death cell were assessed by flow cytometry,cell proliferation capacity was determined by MTS assay,and autophagosome was detected by transmission electron microscopy.Results For the constructed eukaryocyte expression recombinant plasmid pEGFP-DRAM,DNA sequencing showed that the sequence was identical to that of human DRAM cDNA in GenBank.LSCM demonstrated that cells transfected with recombinant plasmid had expression of green fluorescent protein.Flow cytometry analysis showed that cell death was significantly higher in HepG2 cells transfected with plasmid pEGFP-DRAM(P0.05).Percentage of cells in G1 phase was significantly lower(P0.05).MTS assay showed that the proliferation capacity of HepG2 cells transfected with plasmid pEGFP-DRAM was significantly higher(P 0.05).Ultrastructure of HepG2 cells transfected with pEGFP-N1 was essentially normal,whereas autophagosome could be seen in cells transfected with pEGFP-DRAM.Conclusions Eukaryotic expression plasmid pEGFP-DRAM is successfully constructed,and could be expressed in HepG2 cells.DRAM has a two-sided effect in tumor cells.In HepG2 cells,DRAM could induce autophagic death,yet on the other side it also could promote cell proliferation.