The Study Of Apoptosis And Molecular Mechanism After Gins2Knocdown In K562and Nb4Cells

Evaluation the interaction between PML-C and GINS2insidecellsObjective: Evaluation the interaction between PML-C and GINS2byYeast two-hybrid and co-immunoprecipitation.Methods: The plasmids of bait-protein and GINS2protein werecotransformaed into yeast AH109, to study the interaction in living cells.Labeled fusion protein eukaryotic expression vectors were constructed andthen cotransfected into human embryo kidney293cells.Co-immunoprecipitation was used to Evaluation the interaction betweenPML-C and GINS2inside cells.Results: Blue clones were found in QDO/X-α-gal plate, eukaryoticexpression vectors were constructed successfully and cotransfected intoHEK293cells, then HA-PML-C protein was immunoprecipitated byanti-HA polyclonal antibody, Myc-GINS2protein was measured bywestern blotting with anti-Myc monoclonal antibody from the immunoprecipitared complex.Conclusion: The interaction between PML-C and GINS2wasidentified by Yeast two-hybrid and co-immunoprecipitation inside cells. Evaluation the interaction between GINS2and PML (NLS-)inside cellsObjective: Evaluation the interaction between PML (NLS-) andGINS2by Immunofluorescence staining and co-immunoprecipitation.Methods: Labeled fusion protein eukaryotic expression vectors wereconstructed and then cotransfected into human embryo kidney293cells.Immunofluorescence staining and Co-immunoprecipitation was used toevaluate the interaction between PML and GINS2inside cellsResults: pCMV-HA-PML(NLS-) and pCMV-Myc-GINS2wereconstructed successfully, HA-PML(NLS-) was tagging by anti-HApolyclonal antibody and FITC-conjugated goat anti-rabbit IgG，Myc-GINS2was marking by anti-Myc monoclonal antibody andTRITC-conjugated goat anti-mouse IgG, the intracellular colocalization of PML(NLS-) and GINS2was detected by indirect immunofluorescencetechnique. Then HA-PML (NLS-) protein was immunoprecipitated byanti-HA polyclonal antibody, Myc-GINS2protein was measured bywestern blotting with anti-Myc monoclonal antibody from theimmunoprecipitared complex.Conclusion: The interaction between PML (NLS-) and GINS2wasidentified by Immunofluorescence staining and co-immunoprecipitationinside cells. THE STUDY OF APOPTOSIS AND MOLECULARMECHANISM AFTER GINS2KNOCDOWN IN K562ANDNB4CELLSObjective: To construct the eukaryotic expression plasmids for siRNAtargeting GINS2gene and to identify its roles about cell apoptosis in K562and NB4cells, which lay the foundation for leukemia research abouttargets and molecular mechanism.Methods: The siRNA sequence targeting GINS2gene wassynthesized and cloned into the plasmid PGPU6/GFP/Neo to form therecombinant vector PGPU6/GFP/Neo-GINS2siRNA. Then therecombinant vector was transfected into K562and NB4cells. The targetgene was detected by QRT-PCR and Western blotting; the apoptotic ratioand cell cycle distribution of K562and NB4cells was examined by flowcytometry. Immunofluorescence staining was performed to locate thecyclinB1in K562cells and NB4cells.Western blotting was used toexamine apoptosis proteins and signal pathway protein.Results: The recombinant vectors PGPU6/GFP/Neo-GINS2siRNAwas successfully constructed. The transcription and translation level ofGINS2in K562NB4cells were significantly inhibited. Flow cytometryshowed apoptotic cells were significant increased compared with control group, and cell cycle arrested at G2phase. Immunofluorescence stainingexamined cyclinB1aggregates in the cytoplasm and this may be a cause ofarrest of cell cycle in G2phase.We also come to a conclusion that GINS2knockdown influence cell cycle through activating p38MAPK.Conclusion: GINS2siRNA can exert potent inhibition on theleukemia cell growth by inducing apoptosis through activating p38MAPkinase...