Inhibitory Effect Of RNA Interferance On VEGF Expression In Pancreatic Carcinoma Cell Line Panc-1

Objective To investigate the expression of vascular endothelial growth factor (VEGF) protein and microvessel density (MVD) in pancreatic carcinoma, and analyze the correlation between VEGF and MVD.To construct VEGF siRNA vector, investigate the inhibitory effect of RNA interference on VEGF gene expression, to lay the foundation for further investigation on the function of VEGF gene in pancreatic carcinoma and for the development of strategy of anti-angiogenesis therapy in pancreatic carcinoma.Methods The immunohistochemical method was used to determine the expression of VEGF and MVD in 22 paraffin embedded samples of pancreatic carcinoma, compared with that in normal pancreas tissues of 22 cases. SiRNA(small interfering RNA, siRNA) for VEGF gene was designed. Two single strand DNA templates were synthesized according to the sequence of the siRNA. When the DNA templates synthesized, two different restriction sites were superimposed, respectively, to the two end of it. The insertion element formed after the DNA templates annealed. Make the blank plasmid linearization by use of restriction enzyme, and then the insertion element was inserted into the blank plasmid by T4 ligase. PCR and sequencing was performed to check whether VEGF siRNA plasmid be constructed successfully or not. Transfect pancreatic carcinoma cell line Panc-1 cells with VEGF siRNA plasmid and negative control plasmid by Lipofectamine 2000 respectively. As a reporter gene, Green Fluorescence Protein(GFP) was evaluated by fluorescent microscopy to estimate the expression efficiencies when the positive cell clones were selected with G418 for 4 weeks. Then positive cell monoclone was selected and cultivatede with G418 for another 4 weeks. After that, GFP was evaluated by flow cytometry and fluorescent microscopy to estimate the expression efficiencies. RQ-PCR(Real-time Quantitative Polymerase Chain Reaction) was used to detect the expression of VEGF mRNA. The expression of VEGF protein was determined by enzyme-linked immunosorbent assay (ELISA) and immunocytochemical stain. The influence to proliferation ability of Panc-1 cells after VEGF RNAi was measured by MTT assay .Results①The percentage of VEGF protein positive expression was 77.3 % in pancreatic carcinoma, while that was 27.3 % in pancreatic tissue. There was statistical significance between the two groups. The average value of MVD was 35.6±3.1 in pancreatic carcinoma, while that was 15.2±2.9 in pancreatic tissue. There was statistical significance between the two groups. The expression of VEGF was positively correlated with the value of MVD closely( r=0.815, P<0.01 ).②PCR and sequencing confirmed that the VEGF siRNA plasmid is successfully constructed.③When transfected by the recombinant plasmid, VEGF gene expression was effectively suppressed and the suppression rate is 77.37%,and the suppression rate of VEGF protein was reach to 44.6%.By immunocytochemical stain, VEGF is strongly positive stained in the control group, while VEGF is weekly positive stained in the group transfected by the recombinant plasmid.④C ompared with control, the group transfected by the recombinant plasmid had a decrease in proliferation rate measured by MTT(P<0.01).Conclusion The expression of VEGF is potentialized in pancreatic carcinoma, and the value of MVD is higher than normal pancreatic tissue, The expression of VEGF is positively correlated with the value of MVD. RQ-PCR can determine the expression of VEGF gene quickly, sensitively and expediently, furthermore, it can avoid pollution. Positive ion liposome Lipofectamine 2000 appears stability,high efficiency and low toxicity in adherent cell transfection. VEGF siRNA plasmid can effectively suppress VEGF gene expression in human pancreatic carcinoma cell line Panc-1.At the same time, the proliferation rate of Panc-1 decreases. All these suggests that VEGF gene could be a target for anti-angiogenesis therapy of pancreatic carcinoma. RNAi is an effective measure for tumor gene therapy. However, to transport siRNA into human cells safely is still an unsolved problem.