Mechanism Of Nuclear Transport Of NLS-RAR?

Background and objective1.NLS-RAR?is obtained by neutrophil elastase?NE?cleavage of promyelocytic leukemia protein-retinoic acid receptor fusion protein?PML-RAR??.In previous experiments,we found that the accumulation of NLS-RAR?in the nucleus in APL cell line and APL patient cells.Therefore,this study mainly explored the nuclear transport mechanism of NLS-RAR?.Method1.Enforced expression of NLS-RAR?in U937 and HL60 cell lines were achieved through a lentivirus vector.Vector group?NC group?and NLS-RAR?group?NR group?were exposure to 1?,25-dihydroxyvitamin D₃?400nmol/L?to induced differentiation in 0,12,24,36,48 hours.The mRNA and protein expression level of CD11b,CD11c,CD14 and CEBP?were measured by quantitative real-time polymerase chain reaction?qRT-PCR?and western blot?WB?.2.The plasma protein and the nucleoprotein were extracted from U937,HL60,NB4 and 293T cells transfected the empty vectors and NLS-RAR?overexpression lentivirus.The localization of NLS-RAR?was detected by western blot and immunofluorescence.3.NLS-RAR?contains the two NLSs from PML and RAR?,respectively.In our study,NLS-RAR?mutated the NLS from PML named mut-1;NLS-RAR?mutated the NLS from RAR?named mut-2;NLS-RAR?mutated the two NLSs named mut-12.The localization of three mutated NLS-RAR?were detected by WB and immunofluorescence.The mRNA and the protein expression of CD11b,CD11c,CD14 and CEBP?were measured by qRT-PCR and WB when U937 cells overexpressing mutated NLS-RAR?dealt with 1?,25?OH?₂D₃.4.The expression of KPNA2 is high in AML cells.The co-localization of NLS-RAR?,KPNA2 and KPNB1 was detected by double immunofluorescent staining.The interaction between NLS-RAR?and KPNA2 was examined by the co-immunoprecipitation.5.U937,HL60,NB4 and 293T cells overexpressing NLS-RAR?was treated with special inhibitors of KPNA2 and KPNB1.WB and IF were used to detect the localization of NLS-RAR?.The siRNA of KPNA2 and KPNB1 was transfected with Lipofectamine 2000 into human embryonic kidkey 293T cells,and the localization of NLS-RAR?was detected by WB.Result1.Based on results of western blot and qRT-PCR,the expression of CD11b,CD11c,CD14 and CEBP?in the NR group has decreased,comparing with that in`the NC group.2.NLS-RAR?was mainly located in the nucleus according to the results of WB and IF.3.The result of WB and IF revealed the localization of NLS- RAR?mutated the NLS of RAR?portion was located in the cytoplasm.The expression of CD11b,CD11c,CD14 and CEBP?had no significant change when NLS-RAR?located in the cytoplasm.4.The result of western blot and co-immunoprecipitation suggested that KPNA2 and KPNB1 interacted with NLS-RAR?.5.Knocking or inhibiting of KPNA2 and KPNB1,NLS-RAR?was transferred from the nucleus to the cytoplasm.Conclusion1.NLS-RAR?is mainly located in the nucleus,and it inhibited the differentiation of AML cells.2.The transport of NLS-RAR?depends on the NLS of RAR?portion.NLS-RAR?located in the cytoplasm has no effect on the differentiation of AML cells.3.NLS-RAR?transports into the nucleus via the KPNA2/KPNB1pathway.