Influence Of EBV Latent Membrane Protein 2A On The Proliferation And Apoptosis Of Human Gastric Cancer Cells

Objective To construct recombinant adenovirus expression vector containing Epstein-Barrvirus(EBV) latent membrane protein 2A(LMP2A) and investigate the biological effects ofLMP2A expression on target cell.Methods The target gene LMP2A was amplified by reverse transcription-polymerase chainreaction (RT-PCR), and the recombinant adenovirus vector carrying LMP2A gene wasconstructed with AdEasy system. The recombinant adenovirus vector pAd-2A was transfectedinto HEK293 cells with lipofectamine 2000 to package recombinant adenovirus, and then gastriccancer cell line SGC-7901 was infected by recombinant adenovirus.The expression of LMP2Aand its effects on the proliferation and apoptosis of SGC-7901 cells were analyzed by RT-PCR, MIT assay, transmission electron microscope(TEM), fluorescence activated cell sorting(FACS)and confocal microscopy.Resultsâ‘ The recombinant adenovirus vector pAd-2A was successfully constructed andidentified by PCR, restrictive enzyme analysis, and DNA sequencing. The recombinantadenovirus vAd-2A packaged by HEK293 cells had stable infectivity. The virus titer was3.16×10¹²pfu/L.â‘¡MTT assays showed that SGC-2A cells had higher proliferation rate thanSGC-GFP cells and SGC cells from 2 days post infection.â‘¢The result of TEM showedultramicrostructure of SGC-2A cells had no obvious change, while SGC-GFP and SGC cellcontrol showed a series of changes and appeared apoptotic bodies.â‘£The result of FACS showedalmost all of the SGC-2A cells expressed cyclin E, part of SGC-GFP and only few of SGC cellcontrol expressed cyclin E.⑤Confocal microscopy analysis showed that the ratio of S-phase cellswas markedly elevated at 48h and 72h after vAd-2A transfaction compared with SGC-GFP andSGC cell control.Conclusion The recombinant adenovirus vector carrying EBV LMP2A gene is constructedsuccessfully, and infective recombinant adenovirus vAd-2A is obtained. The results initiallyproved that LMP2A facilitates the proliferation of SGC-7901 cell and suppresses its apoptosis. Italso suggests the biological effects of LMP2A on target cells may be related to cyclin Eexpression induced by LMP2A in target cells.