Rna Interference Targeting Vegf Studies Of An Experimental Gastric Cancer Treatment

Objective:Vascular endothelial growth factor (VEGF), a crucial promoter of blood vessel growth, was over-expressed in many types of tumor tissue. siRNA targeted to VEGF could effectively suppress the cell proliferation and induce apoptosis in tumor cells. In this study, we applied lentivius-mediated siRNA to inhibit VEGF gene expression in vitro and in vivo, to investigate the feasibility and specificity of gene therapy for gastric cancer, and to explore the chemotherapy sensitivity of gastric cancer treated with paclitaxel.Methods:SGC7901cells were cultured in vitro and divided into three groups:blank control group, VEGF-siRNA-Lv lentivirus infected group and negative Nc-Lv lentivirus infected group, thus three stable transfected cells were established by the GFP detection respectively. Total cell RNA and protein were harvested96h post infection. RT-PCR was used to detect the expression levels of bcl-2and p21mRNA, western blot was used to assess the expression levels of SIRT-1and p53protein. BALB/C nude mice were separated into four groups randomly:blank control group, Nc-Lv negative control group, VEGF-siRNA-Lv group, VEGF-siRNA-Lv plus paclitaxel treated group.2×106/0.2ml cells were injected subcutaneously in the right axillary fossa of each mouse. Once the tumors were emerged, paclitaxel were injected once a week in the VEGF-siRNA-Lv plus paclitaxel treated group. The dimensions of the tumors were measured every three days, and the tumor growth curve were drawn. After2weeks’treatment, the nude mice were sacrisficed, the tumor weight and inhibition rate were evaluated, and the tumors were obtained for pathological examination (HE staining), the mRNA level of vegf、p21、bcl-2were measured by RT-PCR, the protein level of SIRT1、P53、P21、BCL-2、 VEGF、Survivin were measured by Western blot. Immunohistochemistry was performed to detect the expression of CD34protein.Results:96h post infection cells were observed by inverted fluorescent microscope, in the VEGF-s iRNA-Lv lentivirus infected group and the negative Nc-Lv lentivirus infected group, the GFP fluorescence were higher than90%, while in the blank control group, no GFP fluorescence were detected. As compared with the control groups, RT-PCR assay revealed that the expression of bcl-2mRNA was down-regulated significantly (p<0.05), while the expression of p21mRNA was up-regulated (p<0.05) in VEGF-siRNA-Lv infected cells. Western blot indicated that the expression of SIRT1was down-regulated (p<0.05), and the expression of p53was up-regulated (p<0.05) in VEGF-siRNA-Lv infected cells. In vivo, the tumor formation rate in nude mice was100%. The tumor sizes in the blank control group and the negative control group increased significantly, while the growth of the tumor in the VEGF-siRNA-Lv group and VEGF-siRNA-Lv plus paclitaxel treated group were visibly suppressed. As compared with the control group, RT-PCR assay revealed that the expression of VEGF、bcl-2mRNA was down-regulated significantly (p<0.05),while the expression of p21mRNA was up-regulated (p<0.05) in VEGF-siRNA-Lv group. Western blot indicated that the expression of SIRT1、BCL-2、 VEGF、Survivin was down-regulated (p<0.05), and the expression of P53、P21was up-regulated (p<0.05) in VEGF-siRNA-Lv group. Immunohistochemistry proved that the expression of CD34was decreased in VEGF-siRNA-Lv groupConclusions:Lentivius-mediated siRNA not only inhibited VEGF gene and protein expression in SGC7901cells and induced apoptosis by down-regulating the expression of SIRT1, up-regulating the expression of p53which further regulated the transcription of bcl-2and p21, but also suppressed the growth of gastric tumor in nude mice, enhanced the chemosensitivity of paclitaxel to gastric tumor.