The Role Of Evi1/Smad3 In Committed Differentiation Of Leukemia Cells Into Monocytes/Macrophages

Objective:To investigate whether EVI1 affects the differentiation process through recruiting CtBP and HDAC targeting SMAD3 for intervention of TGF?/SMAD signaling pathway after PMA-induced leukemia cells? Methods: CCK8 assay was used to determine the proliferation of target cells.The optimal concentration of PMA drug was selected.K562 cells model were induced by PMA,and morphological changes were observed by Swiss-Giemsa staining.Changes of cell cycle before and after differentiation were detected by flow cytometry.The expression of EVI1,CtBP,HDAC1 and SMAD3 genes was detected by reverse transcription-polymerase chain reaction(RT-PCR).The protein expression of EVI1,Ct BP,HDAC and SMAD3 was detected by immunoblotting.Constructed EVI1 recombinant eukaryotic expression vector pENTER-EVI1,it was transfected into K562 cells,which was performed by Western blotting.The expression of EVI1 protein in K562 cells was detected before and after transfection to determine whether the transfection was successful.Western blotting was used to detect the expression of SMAD3 protein.Results: CCK8 assay had a half IC50 concentration of about 1 ng / ml after 72 h of PMA on K562 cells.PMA-induced K562 cell model was established with 1 ng / ml PMA.Morphological changes were observed by Swiss-Giemsa staining.Compared with the untreated control group,the cells were treated with 1 ng / ml PMA for 72 hours.Suspension growth becomes adherent and accompanied by aggregation growth,cell volume becomes larger,the proportion of cytoplasm increases,the cytoplasm is lighter,the nucleus is reduced,the nuclear ratio is lower,the cell fold increases,the mature single nucleus-the number of macrophages increased,with the direction of differentiation into mononuclear-macrophages.The expression of EVI1,CtBP1,HDAC1 and SMAD3 was detected by reverse transcription-polymerase chain reaction(RT-PCR).The expression of EVI1,CtBP1 and HDAC1 decreased with the prolongation of PMA on K562 cells,and the expression of SMAD3 was significantly increased with the time of PMA(P <0.05).The protein expression of EVI1,CtBP,HDAC and SMAD3 was detected by immunoblotting assay.The results were consistent with the results of reverse transcription-polymerase chain reaction.After transfection,the expression of EVI1 protein was increased and the expression of SMAD3 protein was decreased in K562 cells.Conclusions : PMA can inhibit K562 cell proliferation and promote its differentiation into mononuclear-macrophages.The expression of EVI1,HDAC1 and CtBP1 was decreased after the action of PMA on leukemia cells,and the expression of SMAD3 was increased.After transfection,the expression of EVI1 and the expression of SMAD3 were decreased,indicating that Evi1 might target Smad3 to regulate the differentiation of leukemic cells into monocytes / macrophages.