Effect Of Cyclooxygenases-2 Small Interfering RNA On Gastric Adenocarcinoma Cell Line SG C-7901 In Vitro And In Vivo

AIM:The aim of this study was to investigate the overexpression and co-expression of COX-2 and its related signaling proteins in gastric adenocarcinoma patients,and to find their relationship to gastric carcinogenesis. Cyclooxygenases-2(COX-2)- short interfering RNA is constructed so as to suppress the expression of COX-2 in gastric carcinoma cell SGC-7901.We examined the effects of COX-2 siRNA on growth,apoptosis,cell cycle and invasion of gastric cancer,and the effect of the down-regulation of COX-2 on expression of Prostaglandin E₂(PGE₂)and cancer related genes.Subcutaneous SGC-7901 gastric carcinoma model was established in nude mice.The mechanism of RNAi applying for gene therapy in gastric carcinoma by inhibiting COX-2 in nude mice is investigated.METHODS:The study was divided into 3 parts.1.Immunohistochemical staining was applied to tissue microarray,we examined COX-2,PGE₂,PCNA,VEGF expressions in gastric adenocarcinoma tissue array.2.siRNA targeting COX-2 was transfected into SGC-7901 cells mediated by Oligofectamine.COX-2 mRNA expression were detected by realtime PCR after transfection.The expression of COX-2 and other cancer related genes PCNA, PGE₂,Bcl-2,Cyclin D1 and VEGF was also studied by Western blotting and immunofiuorescence staining after transfection.The phenotypic change of SGC-7901 cells including proliferation,apoptosis and invasive ability after RNAi trasfection was studied by MTT assay,annexinâ…¤staining,flow cytometry, Matrigel 3D growth experiment and Transwell.3.Subcutaneous SGC-7901 gastric carcinoma model model was established in nude mice.Every 4 day the mixture of Oligofectamine and siRNA was injected into the tumors and the tumor volumes were measured.On 30^ndday after the first injection,tumors were resected.The expression of COX-2 and PCNA,CD31, VEGF were studied by immunohistochemistry.Apoptosis in tumors were detected by TUNEL method.RESULTS:1.COX-2 was commonly expressed in gastric adenocarcinoma (68.9%).PGE₂,PCNA,and VEGF(80.0%,84.4%and 73.3%of malignant cells, respectively)overexpression was also detected.The COX-2 expression was elevated with the rising gastric adenocarcinoma stages(χ²=31.855,P＜0.001).The expression of COX-2 in gastric adenocarcinoma was higher than that of the gastric mucosa surrounding carcinomas and normal stomach tissue(χ²=14.231, P=0.027);The result of Spearman rank correlation analysis indicated that the relevance of COX-2,PGE₂,VEGF,PCNA expression was considered to be statistically significant.2.SGC-7901 cells were transfected with siRNA targeting COX-2 mediated by Oligofectamine in vitro.The transfection efficiency was up to over 70%.COX-2 mRNA expression were obviously knocked down after transfection with siRNA. COX-2,PCNA,PGE₂,Bcl-2,Cyclin D1 and VEGF protein expression were also decreased.SGC-7901 cells transfected with siRNA targeting COX-2 showed lowering proliferation activity,decrease of SPF and arresting cell cycle in the G₀/G₁phase.The cell invasive ability was attenuated and cell apoptosis was induced.3.As compared with the control group,the tumors in mice treated with siRNA targeting COX-2 grew slowly and the difference of tumor volumes became significant since the 15th day after the first time of siRNA therapy until the 30^nd day tumors were resected.Meanwhile,cell apoptosis was increased.The expressions of PCNA,CD31 and VEGF were downregulated as shown by immunohistochemistry.CONCLUSION:1.The COX-2,PGE₂,PCNA,VEGF expressions increased with gastric adenocarcinoma histodifferentiation grade rise,which are useful for predicting the degree of malignancy for gastric cancer cells.COX-2 overexpression,via inducing angiogenesis,promoting proliferation,inducting immunosuppression, inducing angiogenesis,may play an important role in gastric carcinogenesis.2.Using RNAi technology,siRNA targeting COX-2 mediated by Oligofectamine efficiently knocks down the expression of COX-2 in human gastric carcinoma cell SGC-7901 cells,inhibits activation of PCNA,PGE₂,Bcl-2,Cyclin D1 and VEGF, results in decrease of cell proliferation activity and invasive ability,arrests cell cycle and induces apoptosis.3.The established subcutaneous SGC-7901 gastric carcinoma models in nude mice are treated with siRNA targeting COX-2.the tumor growth is inhibited and cell apoptosis is induced.The findings of in vivo study is in accordance with those in vitro study.