MiRNA-382-5P Modulates The ATRA-Induced Differentiation Of Acute Promyelocytic Leukemia By Targeting PTEN

ObjectiveTo investigate the effects of miRNA-382-5p and phosphatase and tensin homologue(PTEN)on the differentiation and its' underlying mechanisms in human acute promyelocytic leukemia(APL)NB4 cells.Methods1.APL cell line(NB4)and no-APL cell line(HL-60?THP-1)were exposure to ATRA(1umol/L)to induced differentiation.The protein level of PTEN and granulocytic differentiation marker CD11 b were detected by western blot.2.The potential targets of hsa-miR-382-5p were predicted by TargetScan and PicTar.The miR-382-5p mimics,inhibitors,and negative controls were transfected with Lipofectamine 2000 into NB4 cells respectively.The mRNA expression of miRNA-382-5p and PTEN were measured by quantitative real-time polymerase chain reaction(qRT-PCR).3.PTEN luciferase plasmid carrying the binding site ofmicroRNA-382-5p or mutations in the binding site and miR-382-5p mimics were co-transfected with Lipofectamine 2000 into human embryonic kidney 293 T cells.The effects of miR-382-5p expression on the transcriptional activity of PTEN was examined by luciferase report assay.4.Enforced expression of PTEN in APL cells(NB4)and no-APL cells(HL-60?THP-1)were achieved through a lentivirus vector.Flow cytometry was used to detect cell cycle and myeloid differentiation markers(CD11b?CD14?CD15).Indirect immunofluorescence assay was to observe cellular location and expression of promyelocytic leukemia(PML)and PTEN protein.Results1.Based on the results of western blot and qRT-PCR,we found that cells(NB4?HL-60?THP-1)were all could be induced differentiation by ATRA.However,the expression of miR-382-5p was downregulated by ATRA in NB4 cells.2.Enforced expression of miR-382-5p could inhibit PTEN both in mRNA and protein level,while down-regulation of miR-382-5p increased the expression of PTEN.The potential binding sites of miR-382-5p in human PTEN were predicted by TargetScan and PicTar.The results of luciferase report assay revealed that miR-382-5p expression suppressed PTEN activity at the level of transcription.3.Overexpression PTEN promotes ATRA-induced differentiation ofNB4 cells,not in HL-60 and THP-1 cells.What's more,PTEN sensitives NB4 cells to physiological levels of ATRA and promotes the expression of PML,p53 and HIPK2,thereby restoreing PML nuclear bodies(NBs).4.MiR-382-5p/PTEN axis modulates cell differentiation through regulating cell cycle,mainly effecting the expression of CyclinD1 and G1 phase.Conclusions1.MiR-382-5p modulates the ATRA-induced differentiation of acute promyelocytic leukemia by targeting tumor suppressor PTEN.2.PTEN contributes to the ATRA-induced differentiation of acute promyelocytic leukemia and sensitives NB4 cells to physiological levels of ATRA.3.MiR-382-5p/PTEN axis modulates cell differentiation through regulating cell cycle,mainly effecting the expression of CyclinD1 and G1 phase.4.PTEN promotes the expression of PML,p53 and HIPK2,thereby restoreing PML nuclear bodies(NBs).