A Preliminary Study On The Roleand The Mechanism Of Notch1Regulating NF-κB Signaling In Human Glioma Cell

Objective:1. To detect the effect of Notch1intracellular domain (NICD1)on the activity of NF-κB signaling in human glioma U251cell.2. To compare the effects of NICD1and its derivative peptide△NICD1(1773-2230) on NF-κB signaling activity in human glioma U251cell.3. To detect the influence of NICD1and its derived peptide△NICD1(1773-2230) on the expression levels of NF-κB p65and NF-κB p50.4. To clear the interaction between NCID1or its derivative peptide△NICD1(1773-2230) and the NF-κB p50.Methods:1. Using pNL-IRES2-EGFP-NICD1as a template, acquiring the amplified target gene by PCR and choosing p3xFlag-CMV-10vector as the new vector, two flag-fused eukaryotic expression plasmid vectors p3xFlag-CMV-10-NICD1and p3xFlag-CMV-10△NICD1were constructed.2. Human glioma U251cell was transfected with p3xFlag-CMV-10-NICD1and p3xFlag-CMV-10-△NICD1and then the expression of NICD1and ANICD1in transfected cell was detected by western blotting.3. After U251cell was transfected with p3xFlag-CMV-10-NICD1or p3xFlag-CMV-10-△NICD1, the nuclear protein of NICD1-overexpressed or△NICD1-overexpressed U251cell was extracted and then detected using electrophoretic mobility shift assay(EMSA) to determine the NF-κB target DNA binding activity4. After U251cell was transfected with p3xFlag-CMV-10-NICD1or p3xFlag-CMV-10-△NICD1, the total protein and the nuclear protein of NICD1-overexpressed or△NICD1-overexpressed U251cell was extracted and then detected using western blotting to quantify the expression level of NF-κB p65and NF-κB p50in the whole cell or in the nucleus5. After U251cell was transfected with p3xFlag-CMV-10-NICD1or p3xFlag-CMV-10-△NICD1, choosing NICD1,△NICD1, p65, p50as the stained objects,cell immunofluorescence staining and confocal microscopy detecting was practiced to observe the distribution of p65and p50in cell and intracellular co-localization of NICD1orANICDl with p50.6. After U251cell were transfected p3xFlag-CMV-10-NICD1or p3xFlag-CMV-10-△NICD1, co-immunoprecipitation assay was processed to detect the interaction between Notch1intracellular NICD1or its derivative of peptides△NICD1(1773-2230) and NF-κB p50.Results:1. Tag-fused eukaryotic expression plasmid vector p3xFlag-CMV-10-NICD1and p3xFlag-CMV-10-△NICD1was successfully screened, and furtherly confirmed by restriction digestion, sequencing, expression assy.2. DNA binding activity NF-κB of in3xFlag-NICD1or3xFlag-△NICD1high-expressed U251cell was detected by electrophoretic mobility shift assay (EMSA) and the result shows over-expression of both NICD1and its derivative peptide-segment ANICD1can significantly promote DNA binding activity of NF-κB and ANICD1activation effect seems stronger than NICD1.3. After nuclear protein extracted from U251cell transfected with p3xFlag-CMV-10-NICD1and p3xFlag-CMV-10-△NICD1was detected by Western blot,we found that the level of p50and p65in the nucleus of U251cell of3xFlag-NICD1or3xFlag-△NICD1overexpression significantly increased than the control groups.4. Western Blot analysis showed that p50and p65protein expression significantly increased in NICD1-overexpressed or△NICD1-overexpressed U251cell.5. U251cell overexpressed the NICD1or△NICD1was observed with high p50and p65nuclear transferation and also observed obvious co-localization of NICD1or △NICD1with p50in the nucleus through p50and p65specific immunofluorescence staining.6. Co-immunoprecipitation experiment showed that both NICD1and its derived peptide△NICD1(1773-2230) could interact with NF-κB p50.Conclusions:1. NICD1overexpression in human glioma U251cell can raise the level of expression of p50and p65and strengthen the activity of NF-κB by activating NF-κB and promoting its translocation to nucleus.2. NICD1and NF-κB p50subunit interact directly in human glioma U251cell.3. The derived fragment of NICD1,NICD1(1773-2230), has the similar active effect to NF-κB signaling with intact NICD1.