| Background:Cyclooxgenase-2(COX-2) is a rate-limiting enzyme induced temporarily which catalyze the conversion of arachidonic acid to prostaglandins. There are much evidence which suggest the close relation between COX-2 and tumour especially in alimentary tract .Study showed COX-2 can participate the progression of tumour including invasion and metastasis in many ways. To discover the effect and mechanism of COX-2 in invasion and metastasis is significant in therapy and prevention of tumour. COX-2 may be a possible target to treat the invasion and metastasis of tumour.In order to investigate the effect and mechanism of COX-2 in invasion and metastasis in gastric cancer, we constructed antisense COX-2 eukaryotic expressing vector and then tranfected it into human gastric cancer cell line SGC-7901 with DOTAP liposome firstly. Secondly, we observed the cell's change of the ability in invasion, detected VEGF,VEGF-C and MMP-2 by PCR and Western blot analysis respectively .In order to explore the possibility of COX-2 to act as a new target for cancer therapy, the effect of antisense COX-2 and COX-2 inhibitor on the biological behaviour of SGC-7901 was observed lastly.Objectives:1. To construct and identify an antisense COX-2 gene eukaryotic expressing vector by recombinant technology.2. To investigate the effect and mechanism of COX-2 in the ability of invasion of human gastric cancer cell line SGC-7901.3. To observe the effects of antisense gene of COX-2 and it's inhibitor on the biological behaviour of gastric cancer cell line SGC-7901. To explore the potential value of it in the therapy of gastric cancer. Methods and Results:1. The detection of COX-2 in normal stomach mucosa and gastric cancer by immunohistochemistry.The result showed the expression of COX-2 in gastric cancer is much higher than in normal stomach mucosa. In gastric cancer, the expression of COX-2 is related to the depth of invasion,metastasis in lymph node and metastasis in distance. It has no relation to gender,age,location,size and differentiation. The expression of COX-2 is related to invasion.2. The construction and identification of antisense COX-2 gene eukaryotic expressing vectorFirstly, a 1.8kb fragment of COX-2 cDNA was obtained from the plasmid via EcoRâ… digestion, and added EcoRâ… and BglII site to the two ends of the fragment in two direction inversely. Inserted it into the eukaryotic expressing vector plasmid pIRES2-EGFP by recombinant technique. The resulting antisense recombinant was identified by endonuclease cutting with Pstâ… and DNA sequencing.3. Gene transfection and identification.The transfection of antisense COX-2 recombinant and plasmid pIRES2-EGFP was mediated by liposome named DOTAP. The transfectants were screened by G418 and then idendified by detection of NEO resistant gene with PCR technique. The results showed we obtained stable transfectants.4. The detection of COX-2 in the transfectant.We used PCR and Western Blot technique to determine if the expression of COX-2 was inhibited by antisense COX-2 gene in the stable transfectant. The results suggested that endogenous COX-2 expression could be efficiently down-regulated by transfection of antisense COX-2 gene in cultured SGC-7901 cells.5. The effect of antisense COX-2 on the invasion of SGC-7901.The results of MTT essay,clone formation,Transwell membrane and cell migration showed the transfectant's ability of invasion was reduced.6. The detection of factors related to invasionVEGF,VEGF-C and MMP-2 were detected by PCR and Western Blot. The results showed all these factors related to invasion were down-regulated after transfection.7. The effect of antisense COX-2 and COX-2 inhibitor on the biological behaviour of SGC-7901.The size and weight of transplanted tumor in nude mice were reduced by antisense COX-2 and COX-2 inhibitor respectively. In these transplanted tumor, PCR and Western Blot results showed the expression of VEGF,VEGF-C and MMP-2 were down-regulated.Conclusions:1. The antisense eukaryotic expressing vector of COX-2 was successfully constructed by recombinant technology.2. Antisense COX-2 can inhibit the ability of invasion in SGC-7901 suggested COX-2 can enhance the proliferation and invasion of tumour.3. Antisense COX-2 could down-regulation the expression of VEGF,VEGF-C and MMP-2 suggested COX-2's enhancement of proliferation and invasion may act through these three factors.4. Antisense COX-2 and COX-2 inhibitor can inhibit the transplanted tumor respectively suggested COX-2 inhibitor inhibit the transplanted tumor act through the way of COX-2.5. The expression of VEGF,VEGF-C and MMP-2 were down-regulated in transplanted tumor suggested COX-2 can enhance invasion.6. COX-2 may be a new target in the therapy for human gastric cancer. |
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