[Effects of inhibition of cyclooxygenase-2 by RNA interference on proliferation and apoptosis of human gastric cancer cells: an experimental study with human gastric cancer cells and mice].
OBJECTIVE
To study whether the expression level of cyclooxygenase-2 (COX-2) is correlated with the proliferation and apoptosis of cancer cells and to study whether the RNA interference technique can be used in anti-cancer gene therapy.


METHODS
WBH1, a eukaryotic expression plasmid of shRNA targeting on COX-2, was constructed. Human gastric cancer cells of the line SGC-7901 were cultured and divided into 3 groups: to be transfected with WBH1 or negative control plasmid HK, or used as un-transfected control group. RT-PCR and Western blotting were used to detect the expression of COX-2 mRNA and protein. MTT method was used to detect the proliferation of the cells. The apoptosis of the cells was determined by flow cytometry. Fifteen nude mice were randomly divided into 3 equal groups: 10 to be inoculated subcutaneously with WBH1 plasmid transfected SGC-7901 cells (inhibition group) or negative control plasmid HK transfected SGC-7901 cells, and 5 were used as un-transfected controls. The mice were observed for 4 weeks to observe the survival and the tumorigenesis. Then the mice were killed to take out the tumors. The tumorigenic rate and tumor inhibition rate were evaluated.


RESULTS
The proliferation of the SGC-7091 cells transfected with WHB1 plasmid did not changed significantly 24 and 48 hours after the transfection, however, decreased significantly 96 hours and 1 week after (both P < 0.01). The apoptotic rate of the SGC-7091 cells transfected with WHB1 plasmid was 52.28% +/- 17.91%, significantly higher than that of the cells transfected with the control plasmid HK (0.54% +/- 0.16%) and that of the un-transfected cells (0.52% +/- 0.27%, both P = 0.009) without a significant difference between the latter 2 groups (P = 0.998). Four weeks after inoculation the tumorigenic rate was 100% in both the un-transfected control mice and the mice inoculated with negative plasmid HK transfected SGC-7901 cells. There was no significant difference in tumor size between these 2 groups (P = 0.965). The tumorigenic rate of the mice in the inhibition group was 0.4 with an inhibition rate of 89.8%. The tumor weight of the inhibition group was 0.050 g +/- 0.003 g, significantly lighter than those of the control group and the group inoculated with negative plasmid transfected SGC cells (0.490 g +/- 0.017 g and 0.490 g +/- 0.013 g respectively, both P < 0.01).


CONCLUSIONS
Construction of a eukaryotic expression vector expressing the specific shRNA targeting on COX-2, closely related to the proliferation and apoptosis of tumor cells, and transfection of it into the tumor cells helps inhibit the expression of COX-1, thus inhibiting the growth and proliferation of the tumor cells.
Wang
Bu-hai
BH
Department of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Qian
Wei
W
Gao
Yuan-jun
YJ
Wang
Hua-feng
HF
Hou
Xiao-hua
XH