Expressions of angiopoietin2 in gastric carcinoma and effects of antisense angiopoietin2 cDNA on human gastric carcinoma cell line SGC-7901

Abstract:

Objective：To study the expressions of angiopoietin2（Ang2） in gastric carcinoma tissues and its relationship with pathologi－cal stage and to investigate the biological effects of antisense Ang2 cDNA（pEGFP-N1-anti-Ang2 cDNA） on human gastric carcinoma cell line SGC-7901. Methods：Immunohistochemical method was used to detect expressions of Ang2 protein in 53 samples of gastric carcinoma tissues and Western blot was used to detect expressions of Ang2 protein in 10 samples of gastric carcinoma and 10 normal gastric tissues. Two different plasmids including pEGFP-N1 and pEGFP-N1-antisense Ang2 cDNA were transferred seperately into an in vitro cultured human gastric carcinoma cell line SGC-7901 by using Lipofectamine2000 transfection technique. mRNA and protein expressions of Ang2 after transfection in 53 samples of gastric carcinoma tissue were determined by RT-PCR and immunohisto－chemical method. Effects of antisense Ang2 cDNA on cell viability and apoptosis were determined by MTT assay and flow cytometry（FCM）. The three kinds of gastric carcinoma cells were subcutaneously injected into 30 nude mice and were divided into three groups. Velocity of tumor formation and amount of angiogenesis in tumor tissues were detected. Results：Ang2 protein was positively expressed in gastric carcinoma tissues at different pathological stages（F=166.76，P＜0.000 1），while was unexpressed in normal gastric tissues. Based on RT-PCR and immunohistochemical method，SGC-7901 cell transfected antisense Ang2 gene did not express Ang2 mRNA and its protein. MTT assay demonstrated that the group transfected with antisense Ang2 gene had much lower proliferative capacity than other groups（F=10.39，P=0.002 4）. FCM demonstrated that the group transfected with antisense Ang2 gene also had an decreased cell viability than the other groups（ χ2=3 188.980 5，P＜0.000 1）. Tumor formation was slower in cell transfected with antisense Ang2 gene than in other groups（the 6th d：F=10.18，P=0.000 5；the 18th d：F=7.80，P=0.002 1；the 30th d：F=79.58，P＜0.000 1）. Average number of newly formed vessels was fewer in mice transfected with antisense Ang2 gene than in the other groups（the 1th week：F=15.18，P＜0.000 1；the 2nd week，F=3.50，P=0.044 4；the 3rd week，F=39.02，P＜0.000 1）. Conclusions：Ang2 plays an important role in the angiogenesis and tumor formation. mRNA and protein expression of Ang2 in SGC-7901 cells can be inhibited by antisense Ang2 gene. Antisense Ang2 gene can suppress the in vitro growth of human gastric carcinoma cells as well as the growth and angio－genesis in the human SGC-7901 gastric carcinoma.