The Effect And Possible Mechanism Of Notch1 Gene On Proliferation And Cell Cycle In Human Glioma U251 Cell

Objective:1,To investigate the effect of Notch1 gene on the change of proliferation and cell cycle in human glioma U251 cell.2,To research the possible mechanisms of Notch1 gene involved in proliferation and cell cycle in human glioma U251 cell.Methods:1,The lentiviral vectors, which expressed Notch1 shRNA or Notch1 intracellular domain (NICD), were transfected into U251 cell respectively. RT-PCR and Western Blotting were applied to monitor the validity of down-regulation of Notch1 expression and over-expression of NICD.2,MTT assay was performed to examine the cell proliferation. Flow cytometric analysis was used to detect the cell cycle.3,RT-PCR and Western Blotting were applied to monitor the expression of NF-κB p65. EMSA was performed to assay the NF-κB DNA-binding activity.4,RT-PCR was applied to monitor the expression of capital genes involved in cell cycle, which included p53, p21 , p27 , p16, cyclinD1, cyclinE1, cyclinA2, cyclinB1, cyclinB2, CDK2, CDK4, CDK6, Cdc25C. Then Western Blot was used to check the genes changed in mRNA expression.Results:1,The lentiviral vectors, which expressed Notch1 shRNA or NICD, were efficient in down-regulation of Notch1 expression and over-expression of NICD respectively.2,Down-regulation of Notch1 gene by RNAi inhibited cell proliferation remarkably(P<0.01), arrested cell cycle at G1 phase (P<0.01) and decreased the cell number of S phase(P<0.01). Over-expression of NICD enhanced the cell proliferation significantly (P<0.01), promoted cell cycle at G1 phase (P<0.05) and increased the cell number of S phase (P<0.01).3,Down-regulation of Notch1 gene by RNAi inhibited NF-κB p65 expression(P<0.05) and decreased NF-κB DNA-binding activity(P<0.01). Over-expression of NICD enhanced NF-κB p65 expression (P<0.01) and increased NF-κB DNA-binding activity (P<0.01) in U251 cell.4,Down-regulation of Notch1 gene by RNAi inhibited the expression of cyclinD1, cyclinA2 and Cdc25C (P<0.05). Over-expression of NICD enhanced the expression of cyclinD1, cyclinA2, CDK2 and Cdc25C (P<0.05) in U251 cells.Conclusion:1,Notch1 gene leads to the change of proliferation and cell cycle in human glioma U251 cell.2,Notch1 gene mybe enhance the cell proliferation via increasing NF-κB DNA-binding activity and which may be dependent on increasing the expression of NF-κB p65 partially in U251 cell.3,Notch1 gene maybe enhance the cell cycle via increasing the expression of cyclinD1, cyclinA2, CDK2 and Cdc25C in U251 cell.