NLS-RARΑ Blocks Cell Differentiation By Inhibitng Retinoic Acid Signalling Pathway

ObjectiveTo investigate the effects of NLS-RARα on cell differentiation and its underlying mechanisms of acute promyelocytic leukemia line NB4 cellsMethods1.Enforced expression of NLS-RARα in APL cells(NB4)and no-APL cells(U937)were achieved through a lentivirus vector.NB4 and U937 cells were transfected following the manufacturer’s recommendations.2.Western blot and qRT-PCR were used to mesure the expression of cell differentiation related protein: CD11 b,CEBPβ.3.Western blot and qRT-PCR were used to mesure the expression of classical retinoic acid signalling pathway target genes: RARβ,CEBPε.4.Immunofluorescence analysis and Western blot were used to detect the location of NLS-RARα;Immunofluorescence analysis and Co-IP were used to investigate the space colocation and interaction between NLS-RARα and RA signalling key protein RXRα,NLS-RARα and nuclear corepressor SMRT.5.Luciferase assay was used to investigate the regulation of RARE-luc by NLS-RARα.6.Treated NLS-RARα overexpression cell with ATRA,Western blot was used to detect the expresson of NLS-RARα,CD11 b,RARβ,LIU’s stain was used to observe the cell morphology.Results1.Successful transfection was confirmed using qRT-PCR and Western blot in NB4 and U937 cell.2.Western blot and qRT-PCR analysis revealed that the expression of granulocyte differentiation marker protein CD11 b and CEBPβ were markedly depressed in NLS-RARα over-expression groups comper to the negtive control groups.3.Western blot and qRT-PCR analysis showed that RA signalling target genes RARβ,CEBPε was decreased in NLS-RARα over-expression groups comper to the negtive control groups.4.NLS-RARα primarily located in the nucleus and a small amount was found in the cytoplasm,NLS-RARα colocalize and coimmunoprecipitate with RXRα and SMRT;with the use of ATRA,the amount of NLS-RARα decreased sharply both in the nucleus and cytoplasm,however,the subcellular distribution pattern of NLS-RARα was unchanged;besides,with the use of ATRA,the interaction between NLS-RARα and RXRα,NLS-RARα and SMRT did not disassociate completely;5.In response to ATRA,NLS-RARα exhibits an ATRA-induced transcription activation in a dose-dependent manner;6.ATRA can degrade NLS-RARα protein and induce cell differentiationConclusionNLS-RARα blocks APL cell differentiation by inhibiting the retinoic acid signalling pathway.