RNAi-induced Inhibition Of FLT3 Expression And Induction Of Apoptosis In HL-60 Cells

FLT3 (Fms-like tyrosine kinase-3) is expressed at high levels and plays an important role in AML. FLT3 is the single most frequently mutated gene in AML. The mutations include the internal tandem duplication (ITD) within the juxtamembrane domain and the point mutations within the catalytic domain of the kinase.RNAi is a phenomenon of genetic interference mediated by double strand RNA which can degrade mRNA specifically and effectively and cause post-transcriptional gene silencing. Current research has successfully showed the wide application of RNAi in the silence of some specific oncogenes, shedding a light in the prevention and treatment of human cancers.Objective: In order to observe the effects of RNAi-mediated FLT3 gene silencing in AML cell line HL-60 and the effects on the cell line, and to investigate the feasiabiliy that RNAi technology prevented and treated the AML.Methods: Constructed four recombinant siRNA expression vectors: pGenesil-1-FLT3- ITD, pGenesil-1-FLT3-KDM, pGenesil-1-FLT3-1 and pGenesil-1-FLT3-2. Then transfected with recombinant vector into AML cells HL-60 via electroporation. After HL-60 cells were transfected with recombinant vectors, the interference effects were detected by RT-PCR and Western blot. The apoptosis indices were detected by flow cytometry and MTT.Results: RT-PCR and Western blot show siRNA-FLT3 can inhibit FLT3 mRNA and protein expression (p < 0.05). Flow cytometry and MTT assay show FLT3-siRNA can induce HL-60 cells apoptosis (p < 0.05).Conclutions: Four recombinant siRNA expression vectors can inhibit FLT3 mRNA expression and can induce HL-60 cells apoptosis, and can be used for the animal experiments in vivo successively.HL-60 cell lines can not be transfected by Lipofectamine 2000, Metafectene and Sofast, but can be transfected by electroporation.FLT3 gene plays an important role in AML for a certainty.