Anti-angiogenesis by Lentivirus-Mediated Small Interfering RNA Silencing of Angiopoietin-2 Gene in Pancreatic Carcinoma

Abstract

Angiopoietin-2 (Ang2) has been shown highly expressed in resected human pancreatic carcinoma samples, but the role of it is less clear. We were, therefore, interested in exploring the effects of Ang2 silencing on the angiogenesis and growth of pancreatic carcinoma. Lentivirus mediated Ang2 small hairpin RNA (LV-RNAi) were transfected into pancreatic carcinoma cell line MIA PaCa-2. Three groups were designed in this study: the control group (Mia PaCa-2 cells), the LV-NC group (cells transfected with the control GFP-lentivirus) and the LV-RNAi group (cells transfected with LV-RNAi). The mRNA and protein level of Ang2 gene were detected by real-time polymerase chain reaction and Western blot respectively. MTT assay and Flow Cytometry were used to detect the cell growth and apoptosis. Anti-angiogenesis effect was measured by chick embryo chorioallantoic membrane (CAM) assay. In nude mice bearing tumors, after treatment with intratumoral injection of LV-RNAi, mice growth and tumor volume were observed, and the expression of Ang2, VEGF and CD34 were measured by immunohistochemistry. Compared with the control group and the LV-NC group, the mRNA and protein level of Ang2 gene were successfully knocked down in LV- RNAi group. Also the vessel count was decreased in CAM assay after LV-RNAi transfection. Meanwhile, no obvious cell viability and apoptosis changes were found in MTT assay and Flow Cytometry, respectively. LV-RNAi inhibited pancreatic carcinoma angio- genesis and growth by downregulating the expression of VEGF and CD34. These findings demonstrate that Ang2 gene silencing may exert a anti-angiogenesis effect in vitro and in vivo, and Ang2 targeted gene therapy has the potential to serve as a novel way for pancreatic carcinoma treatment.