Expression Of TOP2A In Gastric Carcinoma And Construction Of TOP2A Low-Expression Model In The Gastric Adenocarcinoma Cell

[Objective]TOP2A is of particular interest with respect to cancer therapeutics, because it is vital in the segregation of newly replicated chromosome pairs,in chromosome condensation,in forming chromosome scaffolds,and in altering DNA superhelicity,and even more importantly,it is also the molecular target for a large group of clinically relevant anti-cancer drugs termed topolI-inhibitors.We found the higher level of TOP2A in the gastric adenocarcinoma according to the gene chip technology.Our research was prepared to identify the distinction of TOP2A in the human gastric carcinoma tissue.Then we can identify the effect between different expression of TOP2A to the biological behaviour of gastric adenocarcinoma cell by constructing low-expression model.It will provide us satisfactory foundation for approaching the prediction effect of the expression of TOP2A to the therapeutic effect.[Methods]1.200 gastric adenocarcinoma cases in 1972-2008 who had immunohistochemistry results ofTopoisomeraseⅡαwere collected and analysised by different expression of TopoisomeraseⅡα/differentiation/position of the tumor, the expression of topoisomeraseⅡαin the MGC-803/BGC-823/MCF-7 cells was detected by WB.2.Construct the recombinant retrovirus plasmid of pSRZTopⅡαand transfect the plamid into packaging cell G3T-hi,collect the virus supematant of packaging cells and detect the virus titer.Infect the target cell BGC-823,MGC-803 and MCF-7 with supernatant and separatethe positive one to incubate.Detect the expression of topoisomeraseⅡαin target cells before after infected by recombinate retrovirus pSRZTopⅡαby WB,and record the difference of cell activity and proliferativeability by cell growth curve.[Result]1.The immunohistochemistry results of 200 cases were all positive, there showed no significant difference in expression of TopoisomeraseⅡαwith sex/age of patients or the differentiation/position of gastric adenocarcinoma.The expression of TOP2A gene found in three adenocarcinoma cells by WB.2.The enzyme digestion and sequencing results showed the construction of plasmid pSRZTopⅡαis succeed.After transfection,collect the supematant and get the highest level of 1.87×10~7 in virus titer.It showed the success of constructing retrovirus.It was obviously that the GFP ratio of cells at the 64h was higher than that at 17h after FACS.So the infection of retrovirus was succeed.It showed lower expression of topoisomeraseⅡαin the target cells than negative control group after infection.The gastric carcinoma cell MGC-803 grew lower after infection and the cell counts in experiment group were lower than negative control group.[Conelusion]1.ShRNA target TOP2A can inhibit the expression of TopoisomeraseⅡαtransiently 2.Inhibition TOP2A gene can inhibit the growth of tumor cells relatively.3.At the first time,the cell model of low expression of topoisomeraseⅡαwas successfully constructed.