Effect Of RNA Interferance On WT1 Gene Expression

Objective To investigate inhibitory effect of RNA interference on WT1 gene expression and construct WT1 siRNA vector to lay the foundation for further investigation on the function of WT1 gene and development of strategy of gene therapy targeting WT1 in leukemias.Methods SiRNAs(small interfering RNA, siRNA) for WT1 gene was designed and synthesized, then be transfected into breast cancer cells line MCF-7 by positive ion liposome Lipofectamine 2000. Transfect effiency was assessed by flow cytometry. Use RQ-PCR(Real-time Quantitative Polymerase Chain Reaction) to detect the expression of WT1 andβ-actin mRNA, and then screening the most effective siRNA. Expression of WT1 protein was detected by western blot. Transfect MCF-7 cells with the most effective WT1 siRNA at different concentration to survey the effect of different concentration WT1 siRNA on RNAi potency. Use the most effective siRNA (10nmol/L)transfect MCF-7 cells, harvest MCF-7 cells at 24,48,72 96 and 120h respectively , and assessed by RQ-PCR to survey how long the WT1 siRNA induced RNAi effect can preserve. The apoptosis induced by VCR which tested in MCF-7 cells was assessed through flow cytometry. Two single strand DNA templates were designed according to the sequence of siRNA which has confirmed to be effective to knockdown WT1 gene. 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. Enzyme cutting, PCR and sequencing was performed to check whether WT1 siRNA plasmid be constructed successfully or not. Transfect K562 cells with WT1 siRNA plasmid and negative control plasmid by Lipofectamine 2000 respectively. As a reporter gene, Green Fluorescence Protein(GFP) was evaluated by flow cytometry and fluorescent microscopy to estimate the transfection efficiencies and expression efficiencies when the positive cell clones were selected with G418. The proliferation ability of leukemia cells was measured by typan blue exclusion assay, MTT assay and colony forming assay. Furthermore, to verify whether knockdown WT1 could induce...