Notch1 Regulates The Stemness And Multidrug Resistance In Human Glioma Cells

Objective:1. To explore the relationship between Notch1 gene and stemness in human glioma U251 cells. 2. To investigate the correlation between Notch1 signal and multidrug resistantance in human glioma cells.Methods:1. U251 cell lines which steadily over-expressed NICD or knock-down of Notch1 gene were obtained by packaging lentivirus, transfection and screening. To be identified through Western Blotting and ICC. 2. The positive rate of CD133+ in the U251 cells was measured by Flow Cytometry. 3. Expressions of Nestin and GFAP in the U251 cells were detected by ICC. 4. The formation rate of tumor spheres in the U251 cells was calculated in stem cell cultures. 5. The tumorigenic ability of the U251 cells with steadily over-expressed NICD was tested in SCID mouse. 6. The cell survival rates of chemotherapy drugs BCNU and VM-26 in the U251 cells were measured by MTT assay.7. The drug efflux function in the U251 cells was detected by Rhodamine123.Results: 1. Successfully obtained U251 cell lines which steadily over-expressed NICD or knock-down of Notch1 gene. 2. The results of Flow Cytometry showed that the positive rate of CD133 in U251 cell lines with over-expression of NICD was higher than that of control group.The positive rate of CD133 in cell lines with Notch1 RNAi was lower than that of control. 3. The results of ICC indicated Nestin were higher expression in U251 cell lines with over-expression of NICD,but GFAP were lower expression. Nestin were lower expression in Notch1 knock-down U251 cell lines compared to controls,but GFAP were higher expression. 4. Experiment results of stem cell culture showed that the formation rate of tumor spheres in U251 cell lines with over-expression of NICD was higher than that of control group.The formation rate of cell lines with Notch1 RNAi was lower than that of control. 5. The result of the tumor formation experiment showed that the tumorigenic ability in U251 cell lines with over-expression of NICD was stronger obviously than the control group. 6. MTT assay showed that the cell survival rates of chemotherapy drugs VM-26(1.5μM) and BCNU(150μM) in cell lines with over-expression NICD were both higher than that of control group. The cell survival rates of cell lines with Notch1 RNAi were both lower than that of control. 7. The results displayed that the drug efflux function in cell lines with over-expression was stronger than that of control group.The drug efflux function of cell lines with Notch1 RNAi was worse than that of control.Conclusions: Notch1 gene involved in regulation of the stemness and multidrug resistantance in human glioma U251 cell. A potential target for Notch1 gene is expected to be the reversal of drug resistance in glioma therapy.