| Objective: Three siRNAs targeting against VEGF of murine were designed by RNA interference (RNAi) technique and then inserted into pSliencer4.1 CMV neo expression vector to investigate the influence of VEGF gene silencing on growth and metastasis of murine colon carcinoma and to explore novel cancer gene therapy.Methods: 1.Three sequences targeting against VEGF were inserted into pSliencer4.1 CMV neo expression vector after screening, designing and synthesizing. The clone was identified by restriction enzyme digestion and DNA sequencing technique. 2. Murine CT-26 colorectal adenocarcinoma cells were divided into 6 groups: group recombinant plasmid V1, group recombinant plasmid V2, group recombinant plasmid V3, group Lipofectamine, group cells and group Scrambled. The recombinant vector was transferred into CT-26 colon carcinoma cells using lipofectamine. Determination of efficiency of transcription and translation of VEGF was performed by semi quantitative RT-PCR, Western blotting and flow cytometry; cell proliferation, cell cycle and cell apoptosis were measured by MTT and flow cytometry. 3. The mice were divided into normal saline group, recombinant plasmid V1 group, recombinant plasmid V2 group and recombinant plasmid V3 group, Subcutaneous animal model were established with murine CT-26 colorectal adenocarcinomal cells, then eukargon expression plasmids were delivered by subcutaneous orthotopic injection in each group. The growth of tumors and mouse weight were observed in each groups. Different treatments were served and all mice were sacrificed after 2 weeks and the subcutaneous tumors were weighed and measured volumes. The expression of VEGF gene and microvessel density (MVD) was detected by immunohistochemical method. Determination of efficiency of transcription and translation of VEGF of tumor tissues was performed by semi quantitative RT-PCR, Western blotting. 4. Mice were randomly divided into 4 groups which had 8 mice in each group, than an experimental model of hepatic metastasis was developed by injection of CT26 colorectal adenocarcinomal cells via spleen. Three different recombinant plasmids are injected intraperitoneally in each group, the control group only received an injection of same amount of saline. The changes of weight of each group were detected. Different treatments were served and all mice were sacrificed after 19 days. The liver and spleen were removed, then weighed and identified by pathology. The numbers of metastatic tumors are counted by stereological method. 5. The mice were divided into normal saline group, recombinant plasmid V1 group, recombinant plasmid V2 group, recombinant plasmid V3 group and sustainable low doses group capecitabine (group X). Subcutaneous animal model were established with murines CT26 colorectal adenocarcinomal cells, then eukargon expression plasmids were delivered by subcutaneous orthotopic injection in each group and group X was delivered by sustainable low doses capecitabine. The volume of tumors and mouse weight were observed in each groups. Different treatments were served and all mice were sacrificed after 3 weeks and the subcutaneous tumors were weighed and measured volumes. The expression of VEGF gene and MVD was detected by immunohistochemical method. Determination of efficiency of transcription and translation of VEGF of tumor tissues was performed by semi quantitative RT-PCR, Western blotting.Results: pSliencer4.1 CMV neo expression vector was successfully constructed and the sequence of constructed recombinant plasmid was correct by restriction enzyme digestion and DNA sequencing. 2. The expressions of VEGF mRNA and protein were decreased markedly than control group by semi quantitative RT-PCR, Western blotting after pSilencer4.1 CMV neo-VEGF1/ VEGF2/ VEGF3-siRNA transfected using Lipofectamine, its difference has statistical meaning (P<0.01), especially in group V1 and V2(P<0.05: group V1 and V2 vs group V3). 3.The growth of CT-26 cells were inhibited significantly in V1,V2,V3 groups than that of group Lipofectamine,cells and Scrambled (P<0.01), especially in group V1 and V2, which difference has notable meaning compared with V3(P<0.05). 4. Flow cytometry: (1)Distribution of cell cycle suggests that the ration of cells in G0/G1 phase increase but in S and G2/M phase decrease according in V1,V2 than group V3,Lipofectamine,cells and Scrambled (P<0.05); (2)Fluorescent detection of CT26 VEGF showed that fluorescence intensity of group V1,V2,V3 decrease markedly than group Lipofectamine,cells and Scrambled (P<0.01); (3)Apoptosis of CT-26 has no statistical significance between six groups(P>0.05). 5. The volume and weight of the tumor in the test group is less than that in the control group(P<0.05); the expression of VEGF gene and MVD in tumor tissue decrease significantly in test group V1,V2,V3 than control group (P<0.01) by immunohistochemical method .The expressions of VEGF mRNA and protein in tumor tissue also decline significantly by semi quantitative RT-PCR and Western blotting in the test group than that in the control group(P<0.01), especially in group V1 and V2, which difference has notable meaning compared with group V0(P<0.05). 6. A mouse model of CT-26 cell with liver metastasis demonstrated that the volume and weight of the livers of mice in the V1,V2,V3 groups is less than that in the control group(P<0.05). The numbers of tumor in the liver are less in the group V1,V2,V3 than that of group V0(P<0.01), especially in group V1,V2(P<0.05 vs group V3). 7. The anti-tumor group of sustainable low doses capecitabine demonstrated that the volume and weight of the tumors in the test group is less than that in the control group (P<0.05). The expressions of VEGF and MVD in tumor tissues,the expressions of VEGF mRNA and protein in tumor tissues also decline significantly by semi quantitative RT-PCR and Western blotting in the group capecitabine than that in the control group(P<0.01).There are the same lower expressions of VEGF and MVD in tumor tissues,lower expressions of VEGF mRNA and protein in the group capecitabine and group V1,V2. However, the higher expression in the all of above has happened in group V3(P<0.05 vs group V1,V2,X).Conclusion: 1. The pSliencer4.1 CMV neo expression vector targeting against VEGF was successfully constructed. 2. The pSilencer4.1 CMV neo-V1/ V2/ V3-siRNA transfecting target cells CT-26 using lipofectamine has taken gene-silencing effect remarkably. 3. The growth and metastasis of tumor-bearing,the tumor angiogenesis,the expressions of VEGF mRNA and protein of tumor tissue were inhibited significantly after delivery of the pSilencer4.1 CMV neo-VEGF1/ VEGF2/ VEGF3-siRNA. There are significant effects in the siRNA which is aimed gene coding region. 4. The effect of siRNA which is aimed gene coding region silencing is equivalent to that of the anti-tumor group of sustainable low doses capecitabine in tumor angiogenesis and the expression of VEGF. 5. RNA interference technology targeting against VEGF might be a novel gene therapy in the future and its clinical application prospect will be fully broad.
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