CNPY2in Acute Promyelocytic Leukemia Ccur In Molecular Mechanism Research

PART ⅠIDENTIFICATION OF THE INTERACTION BETWEEN PML-CAND CNPY2IN VIVO AND IN VITROObjective：To identify the interactions between PML-C and CNPY2via the yeast two-hybrid and co-immunoprecipitation.Method：The two plasmids expressing bait-protein PML-C and targetCNPY2protein were co-transformed into yeast AH109to demonstratetheir interaction in vivo. Tagged fusion protein eukaryotic expressionvectors were constructed and then cotransfected into human embryokidney293(HEK293) cells. Co-immunopreipitation was used toinvestigate their interaction In vitro.Results：Blue clones were found in QDO/X-α-gaplate. Eukaryoticexpression vectors were co-transfected into HEK293cell. HA-PML-Cprotein was immunoprecipitated by anti－HA polyclonal antibody. MYC-CNPY2was detectde by western blot inimmunoprecipitatedpellet.Conclusion：The interaction between the domain of PML withcoiled-coil structure (PML-C) and CNPY2was proved by the yeasttwo-hybrid technique and co-immunoprecipitation technique. PARTⅡIDENTIFICATION OF THE INTERACTION BETWEENMUT-PML AND CNPY2IN VITROObjective： To identify the interaction between mut-PML andCNPY2.Method：HA-tagged fusion protein (pCMV-HA-mut-PML) expressionvector and Myc-tagged fusion protein (pCMV-MYC-CNPY2) expressionvector were respectively constructed, identified, and cotransfected intoHEK293. The interaction was detected by co-immunoprecipitation.Results： Double restriction enzyme digestion showed thatpCMV-HA-mut-PML and pCMV-MYC-CNPY2were successfullyconstructed. Then HA-mut-PML protein was immunoprecipitated byanti-HA polyclonal antibody and the expression of MYC-CNPY2wastested by western blot with anti-Myc monoclonal antibody from immunoprecipitated complex.Conclusion： The interaction between pCMV-HA-mut-PML andpCMV-MYC-CNPY2was existed by co-immunoprecipitation. PART ШEFFECT OF CNPY2GENE EXPRESSION INHIBITION ONPROLIFERATION AND APOPTOSIS OF NB4CELLSObjective： To investigate the effect of cnpy2gene expressioninhibition on proliferation and apoptosis of NB4cells.Method：The recombinant vector pGpu6/GFP/Neo-cnpy2-shRNAand pGpu6/GFP/Neo-NC were respectively constructed by company,Therecombinated vector pGpu6/GFP/Neo-cnpy2-shRNA, negative vectorpGpu6/GFP/Neo-NC and the empty vectorpGpu6/GFP/Neo wererespectively transfected into NB4cells, The mRNA and protein levels oftarget cnpy2gene were detected by RT-PCR and Western blotting, Theeffect of the cnpy2gene expression inhibition on NB4cell proliferationwas detected by MTT, Cell cycle of NB4cells was assayed by flowcytometry, AnnexinⅤ-FITC/PI double-staining results showed that therate of apoptosis of the recombinant plasmid group.Results：The recombinant vector pGpu6/GFP/Neo-cnpy2-shRNAwas successfully bolted by G418, the recombinant vector pGpu6/GFP/Neo-cnpy2-shRNA was transfected into NB4cells,Theexpression of transcription and translation levels of cnpy2gene in NB4cells were significantly declined, MTT results showed that proliferation ofNB4cells was significantly inhibited, Flow cytometry showed apoptoticcells were significant increased compared with control group,and arrestedat S phase.Conclusion：The inhibition of CNPY2gene expression could arrestthe proliferation and promote the apoptosis of NB4cells.