Suppression Of Human Glioblastoma Stem Cells In Nude Mice By SiRNA Notch-1 Gene Therapy

Objective. Short interfering RNA (siRNA) was used to silence Notch-1 gene and blocks it's expression. Our objective was to determine if siRNA targeting Notch-1 would inhibit the formation and growth of tumors in a nude mouse model of human gliobastoma stem cells.Methods. By switching human glioblastoma cells into a defined serum free neural stem cells medium, glioblastoma spheres were isolated. Then, the human glioblastoma spheres were cultured and transfected with Notch-1 siRNA or nonsense siRNA by OligofectamineTM. The glioblastoma spheres were divided into three groups, Notch-1 siRNA transfected group, nonsense siRNA transfected group and control group. Each group's cells would be subcutaneously injected into 5 nude mices. The Nude mice (male, 3-4 weeks of age) were given subcutaneous injections of 0.2 ml of siRNA transfected or normal TJ905 cells suspension at a concentration. of 1×107 cells/ml in the DMEM medium without serum. A week later, when the tumors were palpable, tumors were directly injected with Notch-1 siRNA complex, nonsense siRNA complex, or PBS every four days for 20 days. Tumor sizes were measured every 3 days and calculated by the formula:volume (mm3)=1/2 length×(width)2. After a 20-day follow-up period, mice were sacrificed. Immunohistochemistry was used to determine the expression of Notch-1 gene.Result. Glioblastoma spheres consisted of brain tumor stem cells (BTSCs) expressing CD133 and nestin. Transfect BTSCs with a oligofectamine/siRNA-Notchl complex resulted in tumor growth suppression in nude mice(p<0.05). After siRNA-Notch1 transfection, the expression of Notch-1 gene was decreased in the tumors when compared to the control group(p<0.05).Conclusion. CD133+ and nestin+ BTSCs can be isolated from the primary cultured glioma cells by cultured in serum free neural stem cells medium. After this kind of transfedtion, siRNA targeting Notch-1 significantly inhibits tumor growth in this mouse model of human glioblastoma stem cells. Further investigation is needed to determine optimal dosing and route of delivery.