Effects And Potential Mechanism Of Notch1 Signal On Adhesion And Migration Of Human Glioma U251 Cells

Objective:1. To observe the effect of Notch1 signal on the adhesion and migration of human glioma U251 cells.2. To explore the mechanism of Notch1 Signal on Adhesion and Migration of Human Glioma CellsMethods:1. The lentiviral vectors, which expressed Notch1 shRNA or Notch1 intracellular domain (NICD), were used to transfect into U251 cell respectively. The cell lines which steadily expressed down-regulation of Notch1 and up-regulation of NICD were screened by RT-PCR and Western Blotting.2. Fibronectin assay and Wound-healing assay were applied to obsere the cell adhesion and migration.3. RT-PCR and Western Blotting were performed to detect the expression of N-cadherin,αN-catenin andβ-catenin in each group, and analyze the effect of Notch1 on these genes.4. Collecting the total protein of cells, then examine the interrelationship of N-cadherin,αN-catenin andβ-catenin by Coimmunoprecipitation.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. Through fibronectin assay and Wound-healing assay, the cell adhesion was remarkably increased in Notch1 down-regulation cells, while it was decreased in NICD over-expression cells compared with control cells. The cell migration was reduced in Notch1 down-regulation cells, while it was enhanced in NICD up-regulation cells compared to control cells.3. There is no difference about the expression of RNA and protein of N-cadherin andβ-catenin between Notch1 down-regulation or over-expression cells and control cells. The RNA and protein expression ofαN-catenin in Notch1 down-regulation cells was potently increased, while in NICD over-expression cells was reduced remarkably compared to control cells.4.β-catenin andαN-catenin antibodies were used to precipitate the total protein of U251 cells respectively, and all the other two proteins could be detected by western blotting.Conclusion:1. Notch1 signal is involved in adhesion and migration of Human glioma U251 cells. Down-regulating Notch1 could enhance cell adhesion and decrease cell migration. Up-regulating NICD could weaken cell adhesion and enhance cell migration.2. The Coimmunoprecipitation of cells total protein showed that there is an interrelationship among N-cadherin,αN-catenin andβ-catenin. Notch1 signal may affect cell adhesion and migration through regulating the expression ofαN-catenin which was significant in cadherin/catenin complex.