The Experimental Research On The CpG Island Methylation Of Androgen Receptor Gene Promoters In Leukemia Cells

DNA methylation plays an important role in the organization of gene expression. Today,the research of methylation is mainly focused on gene promoter regions.Androgen receptor(AR) is one of the steroid receptors.Over the last few years,researches of AR's methylation were mainly focused on steroid-dependent neoplasm,but the reports are few on steroid-independent neoplasm.This experiment was designed to detect the methylation status of AR promoter and reveal its abnormal status in leukemia by methylation specific polymerase chain reaction.On the other hand,we treated the leukemia cell lines by the demethylation regent—Aza-2'-deoxycytidine(5-AzaDC),then detected changes in the methylation status of AR gene promotor and the mRNA expression levels of AR gene before and after demethylation.Finally,we analysised the possible mechanism of 5-AzaDC in gene regulation,and clarified the significance of AR promoter methylation on altered AR expression in leukemia.The correlation between AR methylation and AR expression provided theoretical basis that this epigenetic process appeared to be tightly linked to the formation of neoplasm and give a new direction for neoplasm therapy.Methods:1.Leukemia cell lines HL-60,MOLT-4 and Jurkat were Cultured and treated with 5-AzaDC.2.Methylation specific PCR(MSP) was used to detect the CpG island methylation status of AR promoters in the leukemia cell lines and normal WBC.3.Methylation specific PCR(MSP) was used to analyze the CpG island methylation status of AR promoter in the leukemia cell lines after 5-AzaDC treatment.4.RT-PCR was used to measure the mRNA expression levels of AR in the leukemia cell lines before and after 5-AzaDC treatment.5.Immunohistochemistry technology and western blot respectively were used to detect the expression of AR protein in the leukemia cell lines before and after 5-AzaDC treatment. 6.MSP was used to detect the methylation status of AR CpG island in bone marrow of patients with leukemia.Results:1.MSP analysis showed that CpG islands of AR promoter were partly methylated in the three leukemia lines HL-60,MOLT-4 and Jurkat.Whereas in normal WBC samples, only unmethylated bands were observed.2.CpG islands of AR promoters were demethylated after 5-AzaDC treatment.3.RT-PCR analysis showed that AR mRNA were expressed in the three leukemia lines HL-60,MOLT-4 and Jurkat.4.After treated by 5-AzaDC,the expressin of AR mRNA in the three leukemia cell lines increased,compared with that of the cells before 5-Aza CdR treatment.5.Immunohistochemistry technology analysis showed AR protein was mainly expressed in nucleus of the three leukemia cell lines.After treated by 5-AzaDC,both Immunohistochemistry and western blot showed the expression of AR protein increase compared with that of the cells before 5-AzaDC treatment.6.MSP analysis showed CpG islands of AR promoter were methylated in 15 cases of leukemia.3 of 15 had hypermethylation of AR promoter,and 12 of 15 had AR partly methylation.Conclusions:1.CpG islands of AR promoter had abnormal methylation status in the three leukemia lines HL-60,MOLT-4 and Jurkat.But CpG islands of AR promoter in normal WBC samples were unmethylated.The result indicated that DNA methylation may be one of mechanisms in leukemia pathogenesis.2.After 5-AzaDC treatment,the CpG islands of AR promoters were demethylated, which can provide a new direction for the treatment of DNA methylation disorder.3.After 5-AzaDC treatment,the expression of AR mRNA was increased,which demonstrated that demethylated regent—5-AzaDC enhanced the expression of AR gene in transcriptional level.This result indicated that 5-AzaDC may induce AR demethylation to increase the expression of AR mRNA.4.After 5-AzaDC treatment,The expression of AR protein was increased,which demonstrated that 5-AzaDC enhanced the expression of AR gene in translational level. This result indicated that 5-AzaDC may induce AR demethylation to increase the expression of AR protein,which elucidate the noval treatment strategy for leukemia.5.The same methylation status was observed in bone marrow of patients with leukemia.This fact indicated that AR promoter methylation may be a potential marker for leukemia diagnosis.