Effect To Proliferation And Apoptosis Of Human Gastric Cancer Cell Lines SGC-7901 After ShRNA Mediated COX-2 Express Silencing

Part I: Silencing Human COX-2 Gene by Construction of Eukaryotic Expression Vector Expressing the short hairpin RNAObjective: To Specific suppress the expression of human COX-2 gene by construction the Eukaryotic expression plasmid of shRNA. To study whether the technology of RNA interference could be a way of anti-cancer gene therapy.Method: Plasmids containing two different sequences of human COX-2 mRNA coding region were constructed. We gained two plasmids WBH1 and WBH2. Plasmids were identified by enzyme cutting and sequencing. Detect the effect of inhibition by RT-PCT and Western blot after transfect human gastric cancer cell line SGC7901 with liposomes.Result: The expression of human COX-2 gene are suppressed specific and effectively by WBH1. The efficiency of inhibition is 70.42%. Yet WBH2 is not the same.Conclusion: COX-2 expression in human gastric cancer cells could be inhibited significantly by construction of Eukaryotic Expression Vector Expressing the shRNA. The inhibitory effect is highly related with selection of target sequence. Part II: Effect to Proliferation and Apoptosis of Human Gastric Cancer Cell Lines SGC-7901 after COX-2 Express SilencingObjective: To study whether the expression level of COX-2 is correlated with cancer cells' proliferation and apoptosis .To identify whether COX-2 could be a target to cure cancer. To study whether the technology of RNA interference could be a way of anti-cancer gene therapy.Method: Detect the proliferation and apoptosis of cancer cells with MTT and flow cytometry after COX-2 expression be suppressed. Nude mice were divided into 3 groups. 5 cases were involved every group. Group inhibition: Subcutaneous implant gastric cancer cells which COX-2 expression was suppressed. Group control: Subcutaneous implant normal gastric cancer cells. Group sham: Subcutaneous implant gastric cancer cells that were transfected sham plasmid. Evaluate on morphological, histopathology and tumor inhibition rate, after 4 weeks.Result: Cell proliferation are inhibited significantly (p=0.002) and apoptosis are increased obviously compare to control and sham cells(52.28%± 17.91%vs0.52% ± 0.27%vs0.54% ± 0.16% ,p=0.009). In group control and group sham tumor formation can be found in every case. Tumor tissue average weights of these two groups are 0.49 ±0.017g and 0.49 ± 0.013g, while only 2 cases (2/5) can be found in group inhibition. Tumor tissue average weight of group inhibition is 0.05±0.003g. Tumor growth are inhibited significantly (p<0.01) and the inhibition rate is 89.8%.Conclusion: The expression of COX-2 is high related with cancer cells' proliferation and apoptosis and we could suppress cell proliferation and increase apoptosis by inhibit COX-2 gene expression. Part III: In vivo Electroporation-mediated COX-2 Gene Silencing Suppress the Growth of Human Gastric Cancer Cell Lines SGC-7901 in Subcutaneous of Nude MiceObjective: Evaluate the effect of COX-2 gene silencing on growth of human gastric cancer cell lines sgc-7901 in vivo. Verify whether COX-2 could be a target of gene therapy by RNA interference.Method: Make xenograft nude mice model of SGC-7901. Nude mice were divided into 3 groups. 5 cases were involved every group. Group undergoing therapy and group negative control are injected inhibition and sham plasmid into tumor tissue respectively. Meanwhile group blank are injected comparable volume PBS. Evaluate on morphological, histopathology and tumor inhibition rate, one week after electroporation in vivo.Result: Tumor tissue average weight of group undergoing therapy is 0.300 ± 0.056 gram. It is significantly lighter than group negative(0.523± 0.029 gram) and group blank(0.554±0.058 gram), p<0.01. The inhibition rate is 45.84%. Yet there is no significantly deference between group negative control and group blank, p=0.585. Histopathological diference could not be found in each group.Conclusion: The growth of tumor can be suppressed by COX-2 expression inhibition and COX-2 could be a target of related tumor gene therapy through RNA interference.