Study On Methylation Of ID4Gene And Depressive Effect Of Azacitidine In HL-60Cells

Objective: Relapse is an important challenge which is puzzling the treatment of leukemia. Now researchers find that detecting technology on gene mutation can guide treatment. Epigenetics modification plays a key role in the expression of cancer-related genes. DNA methylation which is contained within Epigenetics modification plays an important role in the development of leukemia. Azacitidine is the representative of demethylated medicine, which was approved for the treatment of myelodysplastic syndrome by Food and Drug Administration in2004. While whether azacitidine can reverse the methylation of leukemia cells remains controversial. So in this research, the methylation of inhibitor of DNA binding4(ID4) in Human Acute Myeloid Leukemia cells HL-60, the inhibiting effect of azacitidine on the HL-60cells and the hypermethylated state of ID4gene were tested, to search the new therapeutic target of leukemia and provide laboratory data for treatment of leukemia with demethylated medicine.Methods:Human Acute Myeloid Leukemia cells HL-60were cultivated in vitro. Methylated level of ID4gene in HL-60cells was detected by Methylation Specificity Polymerase Chain Reaction(MS-PCR). HL-60cells was treated with various concentrations of azacitidine and cell inhibitive rate was investigated by CCK8. The change of methylated level of ID4gene in HL-60cells treated with azacitidine was detected by MS-PCR. The expression of ID4gene in HL-60cells treated with various concentrations of azacitidine was also detected by Reverse Transcription Polymerase Chain Reaction(RT-PCR).Results:MS-PCR showed: The ID4gene in HL-60cells presented methylation. CCK8showed: After treating HL-60cells with variors concentrations of azacitidine for48hours, cell inhibitive rate of HL-60cells was11.11%,36.67%,58.89%,78.89%respectively, meanwhile there was a significant different between the groups(P<0.05). MS-PCR showed: ID4gene presented exhaustive methylation in control group, meri-methylation in0.5μM1μM and2μM groups, exhaustive unmethylation in5μM group. RT-PCR showed: Expect0μM and0.5μM group, the quantity of ID4gene in HL-60cells increased gradually with azacitidine concentrations increasing after treated with azacitidine for48h at the concentrations of1μM,2μM and5μM group, which was dose-dependent.Conclusion:1.Hypermethylated state of ID4gene was one mechanism of malignant proliferations in HL-60cells.2.The survival of HL-60cells was inhibited by methyltransferase inhibitor Azacitidine in vitro with a certain dose dependent manners.3.Azacitidine can reverse the hypermethylated state of ID4gene and recover the expression of ID4gene with a certain dose dependent manners.