Studies Of The Interference Of ShRNA On Keap1in Tumor Cell

Part Ⅰ：Expression of Keap1Gene in Tumor CellsObjective：To verify the expression of Keap1in tumor cells.Methods： Real-time PCR was used to determine the expression of suppressor genekeap1in human gastric cancer and prostate cancer tumor.Results：Amplification specificity was shown for the tumor suppressor Keap1. In theresult of Real-time PCR, Keap1value of human gastric cancer and prostate cancer wereless than12in ΔCt, indicating a higher abundance of Keap1expression in humangastric cancer and prostate cancer. Keap1fragment (240bp) was seen in the PCRproducts electrophoresis.Conclusion： Tumor suppressor Keap1widely express in human gastric cancer andprostate cancer cells with high abundance, and is suitable for the knocked-downverification experiment. Part Ⅱ：Construction and packaging of RNAi lentiviral vectorObjective： RNAi lentiviral vector can be used for viral titer after small packaged isconstructed, then four sets of complex values were determined of the infectionlentiviral particles. Optimum efficiency targets for screening can be prepared by RNAilentiviral vector. Methods：According to the mRNA sequence of keap1on GenBank, four valid targetsare designed for the preparation of the double-stranded DNA Oligo (Keap1-shRNA),which will be cloned to GV115lentiviral vectors by using of the technology ofrecombinant DNA, the these clones are identified after being double digestted. Inpackaged lentiviral vector GV115-Keap1-shRNA are transfected to293T cells by themediation of liposome, and the viral titer can be tested after the viral magma becollected by use of the by-hole method.Results：The insertion sequences were proved correct by DNA sequencing, and RNAilentiviral vector is constructed successfully. Recombinant lentiviral vectors arepackaged, the collection of the recombinant virus stock MOI lentiviral particles is3E+8TU/mL.Conclusion：RNAi lentiviral vector is an experimental tool for follow-up experimentsand the basis and preparation, for the next test of screening the best targets, wasprovided by the successfully construction. Part Ⅲ：Screening effective RNAi vectorsObjective：Screening optimum efficiency targetsMethods： Real-Time PCR was used to detected the expression of the target genemRNA.The interference level was evaluated after data was analyzed,then the besttargets were selected.Results：Real-Time PCR results obtained,in human prostate cancer PC3cells, KD1,KD2, KD3group Keap1gene knockdown efficiency reduction higher than the negativecontrol group (P <0.01), the efficiency of gene knock-down Keap1KD4group was notstatistically significance (P>0.05), and KD2group Keap1gene knockdown efficiencyreduction reached75%(P <0.05). Conclusion：Determined Keap1-RNAi-2is the most effective target.