The Research On The Regulation Of Leukemia Cells WT1Gene Expression And DNA Promoter Methylation By Decitabine

Objectives:Research the WT1gene mRNA expression and the DNA promoterregion methylation changes of leukemia cell line U937, K562, leukemiapatient cell, normal bone marrow mononuclear cells before and afterdecitabine, observe the apoptosis of U937cell line, K562cell line, normalbone marrow mononuclear cells with decitabine. Discuss the possibility ofdecitabine demethylation to activate the silencing or low WT1gene afterallogeneic hematopoietic stem cell transplantation, consequently increase thespecific antigen-antibody reaction to kill leukemia cells.Methods:1. Real-time PCR detection of two kinds of leukemia cell line(U937,K562)and normal bone marrow mononuclear cells WT1gene mRNA expression;2. Bisulfite sequencing method(BSP) detection of two kinds of leukemia cellline(U937,K562) and normal bone marrow mononuclear cells WT1genemethylation in the promoter region of the state; Comparison of the WT1genemRNA expression and methylation differences. 3. Flow cytometry to detection of two kinds of leukemia celllines(U937,K562) and normal bone marrow mononuclear cells apoptosis ratewith treated different concentrations of decitabine;4. Compare two types of leukemia cell lines before and after decitabine WT1gene mRNA and methylation changes.5. Collect samples of clinical patients with decitabine as anti-tumor therapy,determination of changes in WT1gene mRNA and methylation.Results:1. In U937cells, K562cells and normal bone marrow mononuclear cells,theWT1gene relative expression of mRNA were negative,（5015±450）copies/10000copies；and（13±10）copies/10000copies.2. The WT1gene promoter region methylation rate in U937, K562, normalbone marrow mononuclear cells were (89±5.9)%,(4±0.2)%,(2±0.1)%.3. Decitabine can induce U937and K562cells death with a time-and-dosedependence, while acting on the normal bone marrow mononuclear cellapoptosis rate is low comparatively, and did not reflect the time dependence.4. By the different concentrations of decitabine, U937, K562cells WT1generelative expression of mRNA rise with a time-and-dose dependence,especially in U937; U937cell WT1gene promoter region methylation ratesignificantly decreased than before treatment, WT1gene promotermethylation levels before and after of K562get no significant changes. 5. After four patients using decitabine as anti-tumor therapy, WT1genemRNA expression levels rise, and the methylation of promoter region has adifferent level of decline.Conclusions:1. In leukemia cell lines U937and K562, WT1gene mRNA expression andpromoter methylation levels were negatively correlated, whereas thenormal bone marrow mononuclear cells WT1gene expression wasnegative, so as the promoter region hypomethylation.2. Decitabin can induce leukemia cell lines U937and K562apoptosis, and itspro-apoptotic effects are stronger than normal bone marrow mononuclearcells.3. The demethylation of decitabine for leukemia cells lines U937WT1genepromoter region is significant, and improve the WT1gene mRNAexpression effectively. In clinical patients it shows similar effects.