| AIM The aim of this study was to construct a recombinant adenovirus vector that expresses small hairpin RNA against the COX-2, AKT1 and PI3Kp85 gene and to detect the suppression effect on the expression of COX-2, AKT1 and PI3Kp85 in gastric adenocarcinoma cell SGC-7901. And after COX-2, AKT1 and PI3Kp85 were down-regulated, we examined the effects of COX-2, AKT1 and PI3Kp85 shRNA on the invasion and metastasis of gastric adenocarcinoma, and the effects of the down-regulation of COX-2, AKT1 and PI3Kp85 on expression of MMP-2, MMP-9, TIMP-2 and VEGF. The mechanism of COX-2 and PI3K/AKT signal pathway in the invasion and metastasis of gastric adenocarcinoma is further investigated.METHODS The study was divided into 2 parts.1. AKT1+COX-2+PI3Kp85shRNA template DNA sequence, capable of forming a small hairpin structure was designed. After renaturation, cloned into the linearisation plasmid PEGFP6-1, PGENESIL-2, PEGFP6-4, then construct a plasmid expression vector, which coding three strip of shRNA. Subcloned the AKT1+COX-2+PI3Kp85 shRNA expression frame into adenovirus expression vector. After verification, enclose,amplify and titered, then transfected the SGC7901 cell to detect the transfection efficiency.2. The recombinant adenovirus vector plasmid expression vector which contained short hairpin RNA (shRNA) expression construct targeting COX-2, AKT1 and PI3Kp85 was transfected into human gastric adenocarcinoma SGC7901 cells. Realtime PCR and Western blot analysis were used to detecte COX-2, AKT1 and PI3Kp85 expression. And the expression of MMP-2, MMP-9, TIMP-2 and VEGF were also detected by Western blot when COX-2, AKT1 and PI3Kp85 were downregulated. ELESA was used to detecte the ectocytic density change of MMP-2 and MMP-9.The invasion and metastasis ability of the tumor cells were measured by Scarification Transwell,2-dementinal and 3-dementional matrigel matrix.RESULTS 1. By using directional cloning, homologous recombination techniques, to obtain a plasmid which includes three shRNA sequence targeting COX-2, PI3Kp85 and AKT1. Restriction digest patterns and squencing analysis showed that rAd5-C-A-P adenovirus expression vector was constructed successfully. And the expression vector contained fluorescin expression frame, so we detected the transfection efficiency by observing the expression of fluorescin after transfection. And the transfection efficiency was over 90%.2. Recombinant adenovirus vector rAd5-C-A-P mediated shRNA targeting COX-2, AKT1 and PI3Kp85 dramatically down-regulated their expression in SGC7901 cells. And MMP-2, MMP-9 and VEGF were also downregulated, while TIMP-2 was upregulated. The ectocytic density of MMP-2, and MMP-9 decreased. Scarification indicates that compared with control group and nonsense sequence group the ability of invision was decreased obviously. Transwell showed that the number of cells invading through the matrigel was control group (105±4), nonsense sequence group (102.5±6.4). rAd5-C-A-P transfection group (26.4±4.6) (p<0.001). Cell growth on matrigel matrix showed normal cell appearances and mix together in control group and nonsense sequence group cells, while the cells of rAd5-C-A-P transfection group were detached from the matrix or grew in a scattered clustering patterns, signifying poor cell metastasis activities in 2-D matrigel. The rAd5-C-A-P transfection group cells formed only small aggregates as compared with the control group and nonsense sequence group in 3-D matrigel matrix growth(p<0.001).CONCLUSION1. Recombinant adenovirus vector expression vector targeting COX-2, AKT1 and PI3KP85 was constructed successfully, and it may transfect the tumor cell with high efficiency.2. ShRNA targeting COX-2, AKT1 and PI3KP85 downregulates significantly their expression in a sequence-specific manner, exerted invasion and metastasis inhibition effect on SGC7901 cells in vitro. Consequently, combined gene therapy by targeting COX-2, AKT1 and PI3KP85 would be a new strategy in targeting gene therapy of gastric adenocarcinoma. |
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