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.

Abstract

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.