Suppression of growth of pancreatic cancer cell and expression of vascular endothelial growth factor by gene silencing with RNA interference.

ABSTRACT

OBJECTIVE: To explore the anti-angiogenesis and tumor cell growth suppressive effects resulted from gene silencing by RNAi in BxPC-3 human pancreatic cancer cells. METHODS: The designation and transfection of vascular endothelial growth factor (VEGF)-siRNA lentivirus was carried out in vitro. Real-time PCR and western blot were conducted to measure the expression levels of VEGF mRNA and protein. Flow cytometry was employed to evaluate cell apoptosis and cell death. A lactate dehydrogenase (LDH) assay was used to assess the cytotoxicity of VEGF-siRNA. A 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to picture the cellular growth. For the in vivo study, BxPC-3 cells were injected subcutaneously into nude mice to form xenografts. The mice were divided into three groups according to the intervention used. The control group, the negative control group and the knockdown group of mice were injected with saline, an empty lentivirus vehicle and lentivirus carrying VEGF-siRNA, respectively. None of the mice died during the study. When these mice were killed, the xenografts were collected and the tumor sizes of the different groups were compared. Finally, immunohistochemistry was used to assess the VEGF expression level and microvascular density. RESULTS: After the transfection of VEGF-siRNA lentivirus, the cellular expression of VEGF mRNA decreased to 50% of the control and the VEGF protein in the BxPC-3 cells decreased to 30% of the control. Apoptosis and cell death increased after transfection of the VEGF-siRNA lentivirus. The LDH assay showed high cytotoxicity induced by VEGF-siRNA lentivirus transfection. The MTT assay showed slower cellular growth in the knockdown cells. Tumor growth suppression was observed in nude mice that had received the VEGF-siRNA lentivirus transfection, and the tumor sizes of the xenografts in this group were clearly smaller than those in other two groups. VEGF expression and microvascular density were significantly decreased. CONCLUSION: Vascular endothelial growth factor gene silencing via VEGF-siRNA can effectively inhibit the production of VEGF and exert an anti-angiogenesis and tumor cell growth suppressive effect both in vitro and in vivo.