Study On Expression And Mechanisms Of Histone Modifying Enzymes In Pediatric Acute Monoblastic Leukemia

Objectives:To explore the histone modifications in pediatric acute monoblastic leukemia(AML-M5),we investigated.the expression profile of histone-modifying enzymes in the Children diagnosed of AML-M5.To provide a preliminary theoretical basis for realization of clinical individualized treatment of AML-M5 in children,we observed the biology features and behaviors and the expression profile of histone modifying enzymes of the leukemia cells before and after treated with histone deacetylase inhibitor(HDACi)LBH589..Methods:1.We evaluated the m RNA expression profile of 85 genes that encode enzymes involved in histone-modification in 27 pediatric AML FAB M5 samples by using novel real-time PCR array.2.CCK8 was used to evaluate the effect on the proliferation of the cell lines(HL-60,SHI-1)were dealed with LBH589.3.NBT test was used to determine the differentiation of HL-60 cells dealed with LBH589.4.Flow cytometric was used to evaluate the effect on the cell cycle of the cell lines(HL-60,SHI-1)dealed with LBH589.5.The two cell lines(HL-60,SHI-1)were dealed with LBH589 for 24 hours,then the rate of apoptosis were detected by flow cytometric.6.The two cell lines(HL-60,SHI-1)were dealed with LBH589 for 24 hours,then we evaluated the m RNA expression profile of 85 genes that encode enzymes involved in histone-modification in these cells.7.Determination of AURKA,NSD1 and PCAF expression by Western-blot in HL-60 cells and SHI-1 cells treated by LBH589.8.CCK-8 assay was used to detect the effect of LBH589 on the proliferation of primary AML-M5 cells in children.The expression of AURKA,NSD1 and PCAF in primary cells treated with LBH589 was determined by Western-blot.Results:1.We obtained a gene cluster consisting of a total of 28 genes(15 up-regulated genes and 13 down-regulated genes).This gene signature revealed up-regulated expression of putative oncogenes GCN5L2,SETD8,KDM5 C,AURKA and AURKB,and downregulated putative tumor suppressor genes(TSGs)EP300,PRMT3,PRMT8 and NOTCH2.2.With the prolongation of time and concentration,the inhibitory effect of LBH589 on leukemia cells was enhanced.The half inhibitory concentration(IC50)of LBH589 on HL-60 cells were 55.55nmol/L,47.41nmol/L and 30.44nmol/L at 24 hours,48 hours,72 hours.However,IC50 of LBH589 on SHI-1 were 75.24nmol/L,64.56nmol/L,54.68nmol/L,respectively.3.The differentiation rate of LBH589 to HL-60 cells was not significantly different from that of the untreated group,but significantly lower than that of the retinoic acid induced group.4.Treated with LBH589,leukemia cells in G0/G1 phase increased significantly,while in the S phase cells decreased.The proliferation of leukemia cells were blocked by LBH589.5.Treated with LBH589 at 25nmol/L for 24 hours,the apoptosis rates of leukemia cells were higher than that of the control group,significantly.6.The expression of KAT6 B,SETD7,SUV39H1,AURKA,NSD1 and PCAF were significantly upregulated in leukemia cells(HL-60,SHI-1)after treated with LBH589.Meanahile,the expression of HDAC11,DZIP3,CIITA were significantly downregulated.7.The expressions of protein of AURKA,PCAF,NSD1 decreased significantly in leukemia cells treated with LBH589.8.IC50 of LBH589 of the primary leukemia cells from 3 children with AML-M5 were 93.32nmol/L,92.591nmol/L,36.71nmol/L,respectively.Similarly,the expressions of protein of AURKA,PCAF,NSD1 decreased in primary leukemia cells treated with LBH589.Conclusions1.We found that 15 genes were up-regulated and 13 genes were down-regulated in pediatric AML-M5.Oncogenes such as GCN5L2,SETD8,KDM5 C,AURKA,and AURKB were up-regulated and putative tumor suppressor genes EP300,PRMT3,PRMT8,and NOTCH2,were down-regulated.Future studies should investigate whether the alterations in these genes can serve as prognostic markers for pediatric AML-M5.Our findings reinforce the understanding that aberrant molecular pathways underlie the pathogenesis of pediatric AML-M5.Elucidation of these pathways may provide novel therapeutic targets and eventually facilitate the development of personalized treatment for pediatric AML-M5.2.LBH589 can inhibit the proliferation of HL-60 cells,SHI-1 cells and primary cells from children with AML-M5.But the differentiation of HL-60 cells can not be induced by LBH589.LBH589 can block the proliferation of leukemia cells and induce apoptosis in leukemia cells.3.LBH589 can change the expression of leukemia cells in a variety of histone modifying enzymes,suggesting the presence of interaction and interplay between histone modifications.LBH589 can downregulation the expression of AURKA,NSD1 and PCAF in leukemia cells.These genes play an important role in the occurrence,development and prognosis of leukemia.These finds explore new ideas for the further studies of the mechanism of LBH589 on the leukemia cell in children with AML.This study can also laid a theoretical foundation for the exploration of individualized therapy in children with AML.