Notch1-DLL1 Feedback Loop In U251MG Glioma Cell Line

Research background:Notch gene encode single stranded transmembrane receptors ,including Notch 1-4.Notch signaling pathway plays wide and variegated roles in cell growth and development, including switching on and off cell cycle, regulating cell proliferation, differentiation, apoptosis and so on. Recent researches showed that Notch1, its ligands and downstream over-expressed in glioma cell lines and tissues, and were invoved in the regulations of glioma proliferation and apoptosis.Research objection:The expression of Notch1 receptor in glioma and its biological function and mechanism have been reported gradually by more and more studies.But little was reported about the expression of Notch1 ligand and its biological function , especially its regulation mechanism.This study was focused on finding the regulation mechanism of DLL1 , analyzing its biological effect and with the hope of discovering the potential joint biotherapy target point.Research Methods: Up-regulating Notch1 receptors by Notch1 expression plasmid transfection: 48 hours after transfection of Notch1 plasmid, the total protein of U251MG cells was extracted and used to analyze the expression of Notch1 receptor and DLL1 ligand.Blocking Notch1 receptors by Notch1 antibody: Notch1 receptors were obstructed by Notch1 antibody and corresponding secondary antibody. The effect of obstruction of Notch1 receptors was verified by immunofluorescence and the expression level of DLL1 was checked by western blot analysis and real-time fluorescence quantitative PCR (Real time RT-PCR).Biological effect research: The effect of proliferation and apoptosis of different treatment on U251MG cells were evaluated via Flow cytometric apoptosis assay and WST-1 cell proliferation assay.Research Results:Notch1 expression plasmid induced more expression of DLL1 in U251MG glioma cell lines when up-regulating Notch1 receptor.Blocking Notch1 receptors down-regulated the expression of DLL1.Both blocking Notch1 receptors and down-regulating DLL1 induced U251MG cell apoptosis and proliferation inhibition, and jointing two treatments produced more effect than the sum of single treatment.Research Conclusions:In U251MG cells, Notch1 signaling pathway feedback regulates the expression of DLL1. There was a potential positive feedback loop between Notch1 and DLL1, which may become effective joint therapeutic target point.