title: Knockdown of angiopoietin-2 suppresses metastasis in human pancreatic carcinoma by reduced matrix metalloproteinase-2.
abstract: Angiopoietin-2 (Ang2) has been shown highly expressed in resected human pancreatic carcinoma samples, and has tightly combination with tumor angiogenesis, but the role in metastasis of it is less clear. We were, therefore, interested in exploring the effects of Ang2 silencing on the invasion and metastasis of pancreatic carcinoma. Lentivirus (LV)-mediated Ang2 small hairpin RNA (LV-RNAi) and mock lentivirus (LV-NC) were transfected into pancreatic carcinoma cell line MIA PaCa-2. Groups were designed in this study: the control group (MIA PaCa-2 cells), the LV-NC group (cells transfected with the LV-NC), the LV-RNAi-KD1 group (cells transfected with LV-RNAi of knock down sequence (1) and the LV-RNAi-KD2 group (cells transfected with LV-RNAi of knock down sequence (2). Boyden chamber transwell assay was used to detect the cell invasion change. The protein levels of Ang2, MMP-2, and MMP-9 gene and mRNA level of MMP-2, MMP-9 were detected by Western blot and real-time polymerase chain reaction, respectively. Orthotopic pancreatic carcinoma xenotransplantation model were successfully built with MIA PaCa-2 cells injection. After treatment with intraperitoneal injection of LV-RNAi-KD2 (LV-RNAi), mice growth, liver function test, tumor volume and peritoneal metastatic numbers were observed and counted. Moreover, expression of Ang2, MMP-2, MMP-9 were measured by immunohistochemistry. Ang2 expression were successfully knocked down in two LV-RNAi groups, especially in the LV-RNAi-KD2group. Compared with the control group and the LV-NC group, the mRNA and protein level of MMP-2 gene were downregulated significantly in LV-RNAi groups, also the invasion cell number decreased in boyden chamber transwell assay after LV-RNAi transfection. Meanwhile, no obvious MMP-9 gene expression changes were found among all the groups. LV-RNAi injection inhibited pancreatic carcinoma metastasis and growth in vivo by downregulating the expression of MMP-2 not MMP-9. Most importantly, LV-mediated gene therapy with Ang2 knockdown exhibited almost no toxicity in vivo. These findings demonstrate that Ang2 gene silencing exert an anti-metastasis effect in vitro and in vivo, and Ang2 targeted gene therapy has the potential to serve as a novel way for pancreatic carcinoma treatment.
authors.affiliation: Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, China.
authors.forename: Zi-Xiang
authors.initials: ZX
authors.lastname: Zhang
authors.forename: Jin
authors.initials: J
authors.lastname: Zhou
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Dong-Ming
authors.initials: DM
authors.lastname: Zhu
authors.forename: De-Chun
authors.initials: DC
authors.lastname: Li
authors.forename: Hua
authors.initials: H
authors.lastname: Zhao
mesh_terms: D042702:Angiopoietin-2 / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D020778:Matrix Metalloproteinase 2 / Q000235:genetics* / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009362:Neoplasm Metastasis / Q000235:genetics / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection
chemical_list: D042702:Angiopoietin-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9