The Role of RANBP9in Acute Promyelocytic Leukemia Pathogenesis
|School||Chongqing Medical University|
|Course||Clinical Laboratory Science|
|Keywords||Leukemia protein-protein interactions co-immunoprecipitationyeast two-hybrid co-immunoprecipitation protein–protein interactionsLeukemia co-immunoprecipitationpromyelocytic leukemia cells apotosis RANBP9|
PART ⅠIDENTIFICATION OF PROTEIN-PROTEININTERACTION BETWEEN COILED-COIL REGION OFPROMYELOCYTIC LEUKEMIA AND RANBP9Objective：To demonstrate the interaction between coiled-coil regionof promyelocytic leukemia (PML-C) and Ran binding protein9(RANBP9)in vitro.Methods: Fragments PML-C and RANBP9were constructed intoeukaryotic expression vector pCMV-HA and pCMV-Myc separately andthe recombinants were co-transfected into HEK293cells. The interaction ofthem in HEK293was detected through co-immunoprecipitation andwestern blot analysis.Results: In positive group, fusion protein Myc-RANBP9was detectedby western blot with anti-Myc monoclonal antibody afterco-immunoprecipitaion with anti-HA polyclonal antibody.Conclusion: The interaction between PML-C and RANBP9in vitrowas identified by co-immunoprecipitation. PARTⅡSPRY DOMAIN OF RANBP9MEDIATES INTERACTIONWITH COILED-COIL DOMAIN OF PROMYELOCYTICLEUKEMIAObjective: To find the domain of RANBP9involved in interactingwith PML-C.Methods: Plasmids pACT2-SPRY and pACT2-LisH-CTLH wereconstructed. Vectors pACT2-SPRY and pGBKT7-PML-C wereco-transformed into AH109. pACT2-LisH-CTLH and pGBKT7-PML-Cwere co-transformed into AH109.The results were detected by observingblue colony after the transformed cells were cultured for3-5days;Eukaryotic expression vector pCMV-Myc-SPRY andpCMV-Myc-LisH-CTLH were constructed which were co-transfectedwith pCMV-HA-PML-C into human embryonic kidney293cells (inHEK293)respectively. The results were detected byco-immunoprecipitation and western blot.Results: In yeast two-hybrid experiments, blue colonies wereobserved in the plate in which AH109were co-transformed with pGBKT7-PML-C and pACT2-SPRY, while blue colonies were notobserved in the plate in which AH109were co-transformed withpACT2-LisH-CTLH and pGBKT7; Fusion protein Myc-SPRY wasdetected by western blot after co-immunoprecipitation for cellsco-transfected with pCMV-Myc-SPRY and pCMV-HA-PML-C, whileFusion protein Myc-LisH-CTLH was not detected by western blot afterco-immunoprecipitation for cells co-transfected withpCMV-Myc-LisH-CTLH and pCMV-HA-PML-C.Conclusion: The SPRY domain of RANBP9interacting with PML-Cwas demonstrated by yeast two-hybrid system and co-immunoprecipitation. PART ⅢIDENTIFICATION OF PROTEIN-PROTEININTERACTION BETWEEN PML-NLS-AND RANBP9Objective: To demonstrate the interaction between mutantpromyelocytic leukemia (PML-NLS-) and RAN binding protein9(RANBP9).Methods: Eukaryotic expression vector pCMV-HA-PML-NLS-andpCMV-Myc-RANBP9were constructed separately and the recombinantswere co-transfected into HEK293cells afterward. The interaction of them in HEK293was detected through co-immunoprecipitation and western blotanalysis.Results: Fusion protein Myc-RANBP9was detected by western blotwith anti-Myc monoclonal antibody after co-immunoprecipitaion withanti-HA polyclonal antibody.Conclusion: The interaction between PML-NLS-and RANBP9invitro was identified by co-immunoprecipitation. PART ⅣOVEREXPRESSION OF RANBP9INDUCES APOPTOSISIN ACUTE PROMYELOCYTIC LEUKEMIA CELLSObjective: To research the impact of RANBP9on apoptosis in acutepromyelocytic leukemia cells.Methods: The eukaryotic expression vector pcDNA3.1-RANBP9wasconstructed and was transfected into NB4cells afterwards. The apoptosisof NB4was detected by FCM. Caspase-3activity was measured bycolorimetric assay using Caspase-3substrate. The protein levels of Bcl-2and Bax were determined by western blot.Results: The apoptosis rate of NB4transfected with pcDNA3.1-RANBP9was increased. The Caspase-3activity was inducedstrongly in NB4cells which transfected with pcDNA3.1-RANBP9. TheBax protein levels were upregulated, while the Bcl-2protein levels weredownregulated after overexpression of RANBP9.Conclusion: The apoptosis of acute promyelocytic leukemia cellsmay be induced by overexpression of RANBP9through the regulation offactors involved in the mitochondrial apoptotic pathway.