A study of the suppressive effect on human pancreatic adenocarcinoma             cell proliferation and angiogenesis by stable plasmid-based siRNA silencing of             c-Src gene expression.
The non-receptor protein tyrosine kinase c-Src regulates diverse biological             processes by associating with multiple signaling and structural molecules. Overexpression             of c-Src occurs in pancreatic cancer and can be predictive of poor prognosis.             The aim of this study was to investigate the inhibitory effects of plasmid-based             siRNAs targeting the human c-Src gene on proliferation and angiogenesis in the             human pancreatic adenocarcinoma cell line Panc-1. Three siRNAs targeting the c-Src             gene were transfected into the Panc-1 pancreatic adenocarcinoma cell line mediated             by Lipofectamine. Transfection efficiency was assessed by flow cytometry. Real-time             quantitative PCR (RQ-PCR) was employed to detect the expression of c-Src mRNA,             and the most effective siRNA was chosen to be cloned into a plasmid. Two single-strand             DNA templates were designed according to the most effective siRNA sequences. The             short hairpin RNA (shRNA) plasmid targeting c-Src with pGPU6/green fluorescent             protein (GFP)/Neo vector psiRNA-c-Src was constructed. Sequencing was performed             to check whether the plasmid was constructed correctly. Panc-1 cells were transfected             with psiRNA-c-Src and the negative control plasmid (psiRNA-N), respectively. Following             selection with G418, the transfected monoclonal cells were chosen. GFP was evaluated             by flow cytometry and fluorescence microscopy to estimate transfection efficiency.             RQ-PCR and western blotting were used to detect c-Src silencing efficiency. To             verify the effects of gemcitabine chemoresistance of c-Src expression, MTT assay             was performed. ELISA was used to determine VEGF levels in culture supernatants.             In a nude mouse model, tumor growth was studied, c-Src, VEGF expression and microvessel             density in tumor tissue were measured by immunohistochemistry. The transfection             efficiency of siRNA in the Panc-1 cell line was above 90%, the most effective             siRNA could suppress expression of the c-Src gene with an inhibition efficiency             of 86.1%. Sequencing confirmed that the c-Src siRNA plasmid was successfully constructed.             MTT assay indicated that the effect of gemcitabine-induced cytotoxicity was markedly             increased in the psiRNA-c-Src group (P<0.05). Meanwhile, the expression of             VEGF in vitro was reduced significantly (P<0.05) in the psiRNA-c-Src group.             In nude mice bearing tumors, c-Src, VEGF expression and MVD were decreased in             tumors produced from psiRNA-c-Src transfected cells (P<0.05). In summary, the             siRNA expression constructs targeting c-Src could specifically suppress c-Src             expression, inhibit VEGF expression, inhibit cell proliferation and enhance gemcitabine             chemosensitivity in vitro. C-Src gene silencingwas able to inhibit angiogenesis             of tumors in vivo. These findings demonstrate that the c-Src targeting gene silencing             approach has the potential to serve as a novel tool for pancreatic carcinoma treatment.
Department of General Surgery, The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Soochow University, Suzhou, PR China.
Xin
X
Zhao
De-Chun
DC
Li
Hua
H
Zhao
Zhi
Z
Li
Jian-Xin
JX
Wang
Dong-Ming
DM
Zhu
Jin
J
Zhou
Jian-Nong
JN
Cen
D000230:Adenocarcinoma / Q000098:blood supply / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D000818:Animals; D001483:Base Sequence; D048708:Cell Growth Processes / Q000235:genetics; D045744:Cell Line, Tumor; D020868:Gene Silencing; D016391:Genes, src; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000098:blood supply / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D014162:Transfection / Q000379:methods; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse