| Objective To study the inhibitory effect of RNA interference (siRNA) on glioblastoma (GBM) cancer stem cells Notch-1 gene expression in addition to the apoptotic induction.Methods GBM cancer stem cell spheres isolation and culture with serum-free medium (SFM), cancer stem cell neurospheres were next assessed by examining the types of molecular markers CD 133 and nestine via immunocytochemistry.Three Small interference RNAs (siRNAs) targeting Notch-1 gene named siRNA1, siRNA2, siRNA3 were synthesized chemically in vitro with gene bank BLAST. TJ-905 cancer stem cells were transfected twice with the siRNA by using LepofectamineTM2000. The GAPDH siRNA transfected cells, the nontransfected cells and nonspecific siRNAs transfected cells were taken as positive, blank and negative controls. Down-regulation of Notch-1 was demonstrated by real-time RT-PCR and western blot, cell proliferation and apoptosis level was measured by MTT and Annexin V/PI analysis.Results (1)SFM allows for isolation and maintenance of undifferentiated cancer stem cell spheres. Those neurospheres showed immunoreactivity for nestin and for CD 133. (2)SiRNA can be efficiently transfected into TJ-905 cells with LepofectamineTM 2000 by twice transfections. The expression of Notch-1 mRNA in blank, negative controls, siRNA 1,2,3 groups were 1.00±0.05,1.03±0.08, 0.25±0.05,0.42±0.05 0.80±0.02 by real-time RT-PCR scan analysis, The expression of Notch-1 protein in siRNA1 group decreased remarkably. (3) An inhibitory proliferation and a remarkable cell apoptosis can be induced in siRNA 1 transfected group.Conclusions The chemically synthesized siRNA targeted Notch-1 gene could down-regulate the expression of Notch-1 in GBM cancer stem cells. In addition, An inhibitory proliferation and a remarkable cell apoptosis were induced when compared with the control cells in vitro. It was suggested that the suppression of Notch-1 expression and the induction of apoptosis provide a new way to glioma gene therapy.
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