Epigenetic Regulation Of C-KIT By AML-ETO9a In T(8;21) Acute Myeloid Leukemia

Objective: Acute myeloid leukemia (acute myeloid leukemia-AML) is a type of highly heterogeneous aggressive blood diseases, mainly caused by differentiation obstacles, blocked apoptosis and proliferation disorders, t (8; 21) (q22; q22) AML is the most common chromosomal translocation, and after translocation, a fusion gene AML1-ETO is generated. However, the expression of AML1-ETO alone can not lead to the occurrence of leukemia. It shall be coordinated with other disease-causing genetic abnormalities. Epigenetic change play an important role in the development process of t (8; 21) AML. AML1-ETO alternative splicing transcript AML 1-ETO9a can cause leukemia alone, as described in t (8; 21) AML pathogenesis put forward a new theory. This study aimed to elaborate AML1-ET09a regulate key target genes c-KIT by epigenetic change, and participate in the mechanism of occurrence and development t(8;21) AML, thus providing an experimental basis for selective targeted therapy.Methods:In this study, bio-informatics analysis revealed that c-KIT gene upstream of the promoter region contains three AML1 binding sites. Using PCR technology, and we based on genomic DNA as templates, different lengths of c-KIT series promoter gene sequences were amplified. Constructed c-KIT plasmids, respectively, with AML1-ETO and AMLl-ET09a plasmid was co-transfected 293T cells, and we used dual-luciferase reporter gene system technology to deduce the combined site of AML1 on the c-KIT promoter sequence. c-KIT recombinants were mixed with different doses and different ratios of AML 1-ETO and AML1-ETO9a transient, and were co-transfected 293T cells, further comparative study of AML 1-ETO and AML1-ETO9a of c-KIT promoter transcriptional activity regulation were detected by dual-luciferase reporter system. Western blot was used to observe the expression of c-KIT, AML1-ETO after treated with C646, which is an inhibitor of histone acetyltransferase P300. Treated skno-1 and kasumi-1 cells with C646 and at last we use CCK-8, PI staining flow cytometry and giemsa staining to detect P300 influence on cell proliferation, cell apoptosis and cell differentiation.Results:Using PCR technology, c-KIT and AML1-ETO, AMLl-ETO9a recombinant plasmids were constructed and the AML1 binding site on c-KIT gene promoter was inferred that located upstream of the promoter 893bp to 963bp. c-KIT recombinants were mixed with different doses and different ratios of AML1-ETO and AML1-ET09a transient, and were co-transfected 293T cells, dual luciferase assay showed that AML1-ETO, AML1-ET09a fusion protein increase in the number of promoters activity positively correlated, the increase radio of AML1-ETO9a/ AML1-ETO fusion protein is proportional to the promoter activity. When the ratio of AML1-ET09a/AML1-ETO increased to 20:80, the promoter activity increased quickly, and no longer increased with the increased proportion of fusion gene. Western blot showed that after C646 handled with skno-1 and kasumi-1 cells, c-KIT, AML1-ETO protein levels of reduction. Function experiments show C646 can cause AML1-ETO positive leukemia cell proliferation inhibition, apoptosis acceleration and cell differentiation.Conclusion:AML1-ETO9a can combine with the promoter regions of c-KIT. Compared with AMLl-ETO, AML1-ETO9a is easier to bound c-KIT promoter region. Epigenetic modification enzyme P300 can cause the promoter region of c-KIT histone acetylation, increase of the c-KIT expression levels of AML1-ETO positive cell lines, and can promote cell proliferation, inhibit cell apoptosis and cell differentiation. When the ratio of AML1-ETO9a/AML1-ETO increased to 20:80, c-KIT activity increase rapidly, it heralds a rise in malignant cells, and provides experimental theoretical basis for clinical prognosis assessment.