Discovery Of Methylation Silenced Genes In Acute Myeloid Leukemia Cells By Microarray Analysis

Background and Objective:Leukemia is the common hematopoietic malignancy that results from an imbalance between cell proliferation and cell maturation,and the development and progression of leukemia is a multi-factor and multi-step process that involved in genetic and epigenetic changes.DNA methylation is one of the most common epigenetic mechanisms on the regulation of gene expression,and the abnormal methylation genes not only play an important role in the pathogenesis of leukemia,but also provide a specific target for the treatment of leukemia.Therefore,screening for new methylation silenced genes in leukemia can help to reveal its pathogenesis,and provide the important theoretical and scientific basis for the prevention and cure of leukemia.Methods:Acute myelogenous leukemia(AML)cell line HL-60 was used in this study.HL-60 cells were treated by methylation transferase inhibitor 5-aza-2'-deoxycytidine(5-aza-2dC),and then MTT assay,flow cytometry,wright staining and Hochest33342 staining were performed to determine the effects of 5-aza-2dC on the growth,cell cycle, differentiation and apoptosis of HL-60 cells,respectively;To screen for methylation silenced genes in AML leukemia cells,human genome U133 +2.0 chips were used to compare the changes in gene expression profiles following the treatment of 5-aza-2dC in HL-60 cells,RT-PCR was used to validate the results of microarray,and MSP was adopted to detect the methylation levels of gene promoters.Results:1.5-aza-2dC inhibited the growth of HL-60 cells in a concentration-and time-dependent manner,and blocked HL-60 cells at G₂/M phases,induced HL-60 cell apoptosis,enhanced the expression of cell differentiation antigens CD11b in HL-60 cells and induced HL-60 cells to differentiate into mature granulocyte.The most evident effects of differention were in the low drug concentration(0.5μmol/L)of 5-aza-2dC, and the most evident effects of apoptosis were in the high drug concentration(5.0μmol/L).2.A total of 117 differential expression genes were identified by microarray analysis,109 genes were up-regulated after 5-aza-2dC treatment,and 8 genes were down-regulated.The functions of differential expression genes were involved in cell proliferation,apoptosis, cell cycle,toll like receptors signaling pathway and histidine metabolism pathway and etc.3.The expression levels of 12 up-regulated genes(ANXA2,CTNND1,S100A9,S100A8,NQO1,GLIPR1,EPAS1, PRF1,H19,PHLDA1,THBS1,BCSC1)in 5-aza-2dC treated HL-60 cells were confirmed by RT-PCR,which were consistent with the results of microarray.4.The methylation statuses of promoters of 8 validated genes(ANXA2,CTNND1,GLIPR1,EPAS1,PRF1,H19,PHLDA1, THBS1)were detected by MSP.The results showed that after 5-aza-2dC treatment,the un-methylated levels were up-regulated,and the methylated levels were down-regulated in all of the 8 genes,suggesting that the 8 genes were the methylation silenced genes in HL-60 cell line.Conclusion:1.5-aza-2dC can inhibit the growth of HL-60 cells, arrest HL-60 cells at G₂/M phase,induce the differentiation and apoptosis of HL-60 cells,which might be related with the anti-leukemia effects of 5-aza-2dC;2.Microarray analyses were performed to compare the changes in gene expression profiles following the treatment of methylation transferase inhibitor 5-aza-2dC in HL-60 cells,117 differential expressed genes were identified,109 genes were up-regulated, 8 genes were down-regulated;3.8 methylation silenced genes were discovered and validated in HL-60 cell line by microarray analysis and MSP:ANXA2,CTNND1,GLIPR1,EPAS1,PRF1,H19,PHLDA1, THBS1.4.ANXA2,CTNND1,GLIPR1,EPAS1,PRF1,PHLDA1 genes were the first time to report as the methylation silenced genes in leukemia cells in this study.The data can provide the important theoretical and scientific basis for elucidating the pathogenesis of leukemia,and the prevention and cure of leukemia.