Transcriptional Factor Foxo3a Was Related To The Decitabine Induced The MDS Cell Line SKM-1Monocytic Differentiation, Apoptosis,Cell Cycle Arrest And Autophagy

Part I:Effects of decitabine on the SKM-1cell differentiation, cell cycle exist and apoptosis as well as the protein activity of transcriptional factor Foxo3aObjective:This part of the study was to investigate the effects of low-dose decitabine (DAC) on the SKM-1cell differentiation, cell cycle exist and apoptosis. The transcriptional factor Foxoe3a acts as a tumor suppressor, we also detected its activity and the expression level of its target genes in SKM-1cell after DAC treatment.Methods:The membrane differentiation markers CD11b and CD14were evaluated by flow cytometry. The DAC induced cell cycle arrest was detected by PI staining. Cell apoptotic conditions were observed by Hochest33342staining and measured by dual staining of Annexin-V-FITC and PI. Foxo3a protein expression was observed by immunofiuorescence and the activity of Foxo3a was calculated by the protein expression of Foxo3a/p-Foxo3a evaluated by Western Blot. Foxo3a related proteins, such as p21, p27, Bim, FasL and C-MYC were detected by Western Blot.Results:Low-dose DAC could induced the expression of CD14, a monocytic membrane differentiation marker, however, it had no effect on the expression of CD11b, a granulocytic membrane differentiation marker, accompanied by the GO/Gland G2/M cell cycle arrest. Apoptosis was observed after the low-dose DAC treatment, especially the early apoptosis. Protein expression evaluation revealed that DAC could induce the activation of Foxo3a, represented by the increase of the ratio of Foxo3a and p-Foxo3a. The cell cycle related proteins p21and p27were relatively low in SKM-1cell, however, DAC could significantly increase their expression level, and it might contribute to the monocytic differentiation and cell cycle exist after DAC treatment in SKM-1. The expression of mitochondrial pathway related pro-apoptotic protein Bim was apparently induced by DAC in SKM-1cell, but the death receptor pathway related pro-apoptotic protein FasL had no clearly change. Conclusion:DAC could induced the monocytic differentiation in SKM-1cell, accompanied by the cell cycle exist and apoptosis. Foxo3a activation induced by DAC might contribute to this process. Part II:Effects of Foxo3a on the DAC induced SKM-1cell differentiation, cell cycle exists and apoptosisObjective:Investigation the role of Foxo3a in the normal as well as DAC-treated SKM-1differentiation, cell cycle exist and apoptosis and its down target proteins.Methods:In order to transient silence the expression of Foxo3a, SKM-1cell was treated with Foxo3a siRNA or combination with DAC. The differentiation, cell cycle progression and apoptosis were evaluated after different treatment in SKM-1cell. Western Blot were used to reveal the target proteins expression under Foxo3a siRNA or the combination treatment of Foxo3a siRNA with DAC.Results:Transient silence Foxo3a had no apparent effects on the CD14expression, cell cycle progression or apoptosis in SKM-1cell. However, the cell cycle progression related proteins, such as p21and p27, and apoptotic related protein Bim were down-regulated; oncoprotein C-MYC was up-regulated after Foxo3a siRNA. The combination treatment of Foxo3a siRNA with DAC could partly reverse the DAC induced differentiation, cell cycle exist and apoptosis, and accompanied by protein expression change, such as p21, p27and Bim. However, C-MYC had no relation with this process.Conclusion:Activation of Foxo3a may responsible for the DAC induced SKM-1cell differentiation, cell cycle exist and apoptosis. Part â…¢:Effects of Fox3a on the DAC induced autophagy in SKM-1cellObjective:The aim of this part was to investigate the effect of DAC on the autophagy in SKM-1and the role of Foxo3a in this process.Methods:siRNA was used to silence the Foxo3a expression in SKM-1, and the autophagy was evaluated through the expression of essential autophgy protein LC3by western blot, as well as the the autophagy inducer Belcinl and proteins related the autophaosome nucleation and elonagation Atg5, Atg12, Atg16.Results:Silence the expression of Foxo3a in SKM-1could decrease the expression of essential autophgy protein LC3, especially LC3-I, and the proteins related to the elongation of autophaosome and autophagy inducer. DAC treatment could apparently induced autophagy in SKM-1cell, as well as the expression of the Atg5, Atg12, Atg16and Beclin1. Combination of DAC and Foxo3a siRNA treatment in SKM-1cell could reverse the DAC induced autophagy and autophagic-related proteins expression.Conclusion:DAC could induce the autophagy in SKM-1and transcripitional factor Foxo3a might related to this process.