Study On The Aberrant Methylation Of Acute Leukemia Correlative Genes

Acute leukemia is a kind of hematological malignant tumor and threatens people's health and life severely, so to study the etiopathogenesis of leukemia and improve the diagnosis and therapy level is very significant. It is considered that the basic mechanism of tumorigenesis is the derangement of genetics and epigenetics. The term"epigenetics"refers to a heritable change in the pattern of gene expression that is mediated by mechanisms other than alterations in the primary nucleotide sequence of a gene. Following the period of postgenome coming, the researches in epigenetics have become the advancing front of life science. DNA methylation is the important section of epigenetics and plays a substaintial part in leukemia genesis and development. DNA methylation is always taken place in the CpG dinucleotide of CpG islands. The spatial structure of chromosome can be changed by the hypermethylation in gene promoter regions that will lead to transcription inactivation and gene expression silence. The silencing of tumor suppressor genes by the hypermethylation of DNA promoter region is one of the essential pathogenesises of leukemia and this always happens early and frequently. The tumor suppressor genes can be reexpressed through demethylation. The abnor DNA methylation of some gene can be taken as biomarker of leukemia to be used in clinical.ZO-1(zona occludens 1) is a tight junction protein, it belongs to membrane-associated guanylate kinase homologs(MAGUKs). ZO-1 is very essential for maintaining the integrity and polarity of cells and plays an important role in cell junction and signal conduction. Because ZO-1 is homologous to the Drosophila discs-large tumor suppressor protein so it could play a significant role on regulation the proliferation and differentiation of cells. ZO-1 gene is closely correlated with the tumorigenesis and tumor metastasis. Yu Li, et al firstly found the promoter region of ZO-1 gene was methylated in mouse leukemia with RLGS, indicating it might be a leukemia correlative gene. There was still no similar report in human AL.In order to further elucidate the pathogenesis of leukemia and search new acute leukemia(AL)correlative genes, to provide effective methods for early diagnosis, curative effect evaluation, prognosis appraisal, minimal residual disease(MRD)monitoring and molecular therapy target, we studied the abnormal DNA methylation of AL in epigenetics field.At the first section research , we analyzed the methylation status of ZO-1 gene in leukemia cell lines and clinical specimens with MS-PCR,COBRA,bisulfite sequencing and investigated the clinical sense of it. The results showed that the promoter region of ZO-1 was specifically methylated in most kind of AL and no subtype-related profile were observed in different subtype AL. ZO-1 was unmethylated in normal bone marrows (NBM) and nonmalignant hematological diseases. The methylation positive rate was 69.57% in newly diagnosed AL and much higher than that of CR AL(34.78% ). But it was the highest in relapse/refractory AL (91.67%).The results indicated the methylation of ZO-1 gene was closely relative to the leukemia genesis and progressing. It is a new common biomarker for AL. Because of the positive correlation between the methylation status of ZO-1 gene and loading dose of malignant cells in acute leukemia, ZO-1 methylation can be used to monitoring the minimal residual disease. One year survival rate of methylation positive newly diagnosed AL was notably lower than that of negative ones and one year relapse rate of methylation positive patients in either new or CR AL was higher than that of negative ones. Above indicating the aberrant hypermethytion of ZO-1 is a bad prognosis factor for AL. It was also proved in our study that the expression of ZO-1 gene was regulated by the methylation status what will provide a new target for demethylation therapy.With the amelioration of chemotherapy project, especially the emerge of haemopoietic stem cell transplantation, the remission rate and life span of acute leukemia patients have been greatly improved and extended, but there are still so many patients relapsed and died. The minimal residual disease (MRD) is one of the radical reasons of relapse, so setting up a good method to monitoring the MRD is very important. At the second research, we have firstly set up SYBR-GreenI real-time MS-PCR system to quantitively detect the methylation pattern of ZO-1 gene in AL. The dissociation curve was single spike and the relative quotient of standard curve was more than 0.995. The technical system and reaction condition was stable, reproducible, accurate, specific to AL, and there was no non-specific amplification. The sensitivity was as good as to 10-4, one malignant cell could be detected among more than 10000 normal cells. Analyzing the methylation pattern of ZO-1 gene in this way can be worked as a new reliable method for monitoring MRD. Applying this technique in clinical will do good for us to prognose relapse early and make the individualized treatment for AL to be come true.The prognosis of AL has been greatly improved with more and more understanding of the molecular mechanism of AL and targeted therapy , especially the findings of some specific leukemia correlated genes such as BCR/ABL and PML/RARαfusion genes and so on. However, the AL relative genes we have known are so finite as yet, this need us to discover further. The study of abnor modification of DNA methylation will provide us a new pathway to search for new AL correlative genes.To screen more new aberrant methylated leukemia relative genes, we compared methylation profiles of 4 AL to that of NBM with RLGS technique in our third section study. We got more than 300 methylation differential dots. 69 of them were occurred at least in two pairs and 46 dots have got the information of DNA fragments from NotI-EcoRV DNA library. 5 of them have been evaluated by COBRA or MS-PCR and 3 hypermethylated genes(DRD4,FOXF1,BUB3)were conformed specificly in AL. Our research has offered a good groundwork for screening more leukemia correlative genes, searching new leukemia biomarkers and investigating the etiopathogenesis of AL.