The Experimental Study Of Silencing Livin Gene By Transfecting ShRNA Expression Vector Into Pancreatic Cancer Sw1990 Cells With Electroporation

AIM: 1 To optimize the electroporation parameters in sw1990 cells using Green fluorescent protein of Livin shRNA recombinant plasmid vectors as a reporter gene; 2 In this study, Livin shRNA recombinant plasmid vectors were transfected pancreatic cancer sw1990 cells with Electroporation. Influence of Livin gene expression and growth inhibition of sw1990 cells induced by Livin expression vectors was observed in vitro, which will provide an experimental foundation for the further investigation of pancreatic cancer gene therapy.METHODS: 1 pancreatic cancer Sw1990 cell and E.coli DH5αtransfected recombinant plasmid were incubated, and extracted plasmid from E.coli DH5α. 2 The following experiments were divided into four groups: blank control group, pGenesil-1.1-Livin-473 group, pGenesil-1.1-Livin-771 group and pGenesil-1.1-NC group. 3 Plasmid expression vector was transferred into Sw1990 cells by electroporation with differently experimental conditions such as pulse voltage, pulse length and the state of cell vitality.The transfection efficiencies were evaluated by flow cytometry (FCM) and fluorescent microscopy. 4 At 48h, cell apoptosis and relative amount of Livin mRNA were detected to observe Livin gene inhibition effect of sw1990 cells transiently transfected with respective interfering recombinant plasmid. 5 Stable sw1990 cells transfected with respective recombinant plasmid were obtained by G418 selection. Expression of Livin mRNA was estimated by using RT-PCR, FCM and cell proliferation ability was evaluated by cell count.RESULTS: 1 The highest electroporation transfection rate (55.86±2.14)% was achieved under the condition parameters such as pulse voltage 260V, pulse length 20mS and vigorous state of sw1990 cells. 2 The transient experimental results at 48h indicated that the apoptotic index was 4.09 and 3.61 times in pGenesil-1.1-Livin-473 group and pGenesil-1.1-Livin-771 group respectively when compared with blank control group (*P< 0.05) and the inhibition rate of Livin gene on Sw1990 cells was 47.62% and 32.91% respectively compared that of control group (*P<0.05). 3 Stable sw1990 cells transfected with various recombinant plasmids were selected with G418. FCM results showed that the percentage of stable transfection cells was above 97% in each group. The inhibition rate of Livin mRNA was 72.86% and 46.51% in pGenesil-1.1-Livin-473 group and pGenesil-1.1-Livin-771 group respectively. Compared with the control group, statistically significant differences were observed (vs control, *P<0.05). The Comparison results of cell proliferation rate between each groups indicated that the proliferation ability of stably transfected cells was decreased obviously. 4 The results also showed that the interfering effect of pGenesil-1.1-Livin-473 against Livin gene was significantly superior to pGenesil-1.1-Livin-771. The inhibition effect of tumor cells'proliferation in pGenesil-1.1-Livin-473 group was also more obvious than that of Genesil-1-Livin-771 group. No obvious effect of interfering Livin gene was observed between pGenesil-1.1-NC group and control group, and also no significant ffect of sw1990 cell proliferation was found in these groups.CONCLUSION: Optimised parameters of electroporation mediated Livin shRNA recombinant plasmid vectors transfecting pancreatic cancer Sw1990 cells were aquired in this study. Livin shRNA recombinant plasmid vectors pGenesil-1.1-Livin-473 and pGenesil-1.1-Livin-771 could successfully interfer the Livin gene expression of sw1990 cells and inhibit the proliferation of pancreatic cancer SW1990 cells in vitro. 5'-GGAAGAGACUUUGUCCACA-3'and 5'-GCGCCGUGUCCAUCGUCUU-3'could be used as the RNAi targets specific to Livin gene in pancreatic sw1990 cells.