The Study Of The Inhibitory Effect Of SiRNA Targeting Specific Genes On The Growth Of Glioma

[Objective] To study the inhibitory effect of small interference RNAs ( siRNAs) targeting IGF-1R on the growth of glioma ; To construct eukaryotic vectors expressing short hairpin RNA(shRNA) targeting Vascular endothelial growth factor(VEGF) and nerve growth factor(NGF) ,which could be used in the glioma gene therapy .[Methods] Part one and part two : Small interference RNAs ( siRNAs) targeting IGF-1R and IGF-1 were synthesized chemically in vitro. C6 cells stably expressing IGF-1R and IGF-1 were transfected with the siRNA by using oligofectamineTM2000 reagent. siRNA transfection efficiency was detected by fluorescent microscopy . RT-PCR and Western blot were used to analyze the down-regulation of IGF-1R and IGF-1 expression. The invasiveness of C6 cell in vitro was measured by the matrigel invasion assays. Flow cytometry was performed to analyze cell apoptosis. The ability of the tumorigenicity in nude mice was measured.Part three: Specific DNA oligonucleotides targeting VEGF and NGF at different sites in their open reading frames were synthesized and cloned into the downstream of the H1 promoter of pSUPER plasmids linearized by BglⅡ-HindIII to construct recombinant plasmids encoding shRNA.[Results] Part one: Green fluorescence in the cells was seen clearly in FITC-siRNA transfected group under the fluorescent microscope, the transfection efficiency of oligofectamineTM2000 was approximation 100%.The expression of IGF-1R could be suppressed effectively by siRNA targeting IGF-1R. The expression of IGF-1R mRNA decreased by 10% - 80% in the siRNAs transfected C6 cells by RT-PCR scan analysis as compared with that of the controls. The expression of IGF-1R protein decreased by 50%.Cell apoptosis could be induced from 1.14%±0.79% in control group to 22.47%±3.15% in IGF1R-siRNA transfected group. The ability of the tumorigenicity in nude mice was reduced.