A Preliminary Study On The Role And Mechanism Of Notch1 In Regulating Smad2 And Smad3 Signaling Molecules Of The TGF-beta Pathway In Human Glioma Cell

Objective:1. To explore whether there exist relationship between Notch1 and TGF-?signaling pathway in human glioma U251 cells.2. To investigate the mechanism of Notch1 acting on Smad2 and Smad3 signaling molecules of TGF-? pathyway in human glioma U251 cells.Methods:1. U251 cell lines which steadily over-expressed NICD or RNA interference of Notch1 gene were obtained by packaging lentivirus, transfection and screening. The expression of Notch1 in transfected U251 cells was identified through Western blotting.2.The total and nucleus expressions of Smad2 and Smad3 gene in the U251 cells were detected by Western blot.3.Expressions phosphorylation of Smad2?Smad3 in nucleus of the U251 cells were detected by immunofluorescence staining.4.Using electrophoretic mobility shift assay(EMSA) to determine the binding activity between P-Smad3 and its target DNA in the Notch1 RNA interference U251 cells.Results:1. Successfully obtained U251 cell lines which steadily over-expressed NICD or RNA interference of Notch1 gene.2. Western Blot indicated Smad2 and Smad3 were higher expression in U251 cell lines with over-expression of NICD; Smad2 were higher expression in Notch1 RNA interference U251 cell line, Smad3 were lower expression in Notch1 RNA interference U251 cell line compared to controls.With the over expression of NICD inU251 cell,the expression of Smad2 in nucleus had no difference compared to controls,while the expression of Smad3 in nucleus were higher;In the nucleus of Notch1 RNA interference U251 cell line,the expression of Smad2 were lower compared to controls,the expression of Smad3 had no difference compared to controls.3. Immunofluorescence staining showed over expression of NICD and Notch1 RNA interference in U251 cells the total and nucleus expression of P-Smad2 were higher compared to controls.The total and nucleus expression of P-Smad3 had no difference between the over expression of NICD and the control group; with the expression of Notch1 RNA interference in U251 cells the total expression of P-Smad3 were higher compared to control group, while in the nucleus the expression of P-Smad3 were lower compared to control group.4. Binding activity was decreased between P-Smad3 and its target DNA in the Notch1 RNA interference U251 cells.Conclusions1. NICD over expression can promote the expression of Smad3,Notch1 signal down-regulation can inhibit the expression of Smad3.2.The mechanism of Notch1 regulation of Smad3 involved the regulation of phosphorylation activation and its binding activity with the target gene.Downregulation of Notch signaling can promote the phosphorylation activation of Smad3,inhibit the expression of P-Smad3 in the nucleus and the binding activity of DNA to the target.