Gene Expression Regulation Of SIRT1 And EZH1 By AML1-ETO In Acute Myeloid Leukemia

Objective:Acute myeloid leukemia is a clonal aggressive blood disease caused by the differentiation and apoptosis obstacles and uncontrolled proliferation of myeloid cells.The most common chromosomal translocation of AML generating the AML1-ETO fusion gene is t(8;21)(q22;q22).AML1-ETO protein can bind directly with the promoter of downstream gene to up-regulate gene expression,or together with expression product on target gene,it plays key role in abnormal transcriptional regulation of hematopoietic differentiation,proliferation and apoptosis,which is considered to be one of the pathogenesis reason of leukemia.SIRT1 is closely related to hematologic diseases.This study is designed to further analyze the SIRT1 gene expression regulation mechanism,to look for core promoter region of SIRTl,to explore the impact of SIRT1 on AML1-ETO positive leukemia,therefore provide experimental foundation for the development of selective targeting SIRT1 therapy.Epigenetic changes include histone deacetylation and histone methylation, epigenetic disorder is closely related to the development of blood diseases.SIRT1 gene product can deacetylase histone and non-histone to make changes in a number of physiological functions; EZH1 is an important histone methyltransferase, which can lead to histone methylation;therefore, this study was designed to further analyze the expression and regulatory mechanisms of EZH1 and SIRT1 genes,to look for core promoter region of them in hematologic malignancies, to explore the impact of epigenetic changes on the development of AML1-ETO fusion gene-positive leukemia and to provide an experimental basis for selective targeted therapy.Methods:In the first part of the study,the RT-PCR was used to preliminary detect the expression of SIRT1 in AML1-ETO positive leukemia cell line and clinical samples of leukemia patients.A series of possible core promoter fragments of the SIRT1 5’-untranslated region were amplified by PCR,the PCR products were authenticated by XhoⅠ/HindⅢ co-digestion and DNA sequencing and cloned into XhoⅠ/HindⅢ co-digested pGL3-Basic reporter vector,the poly-cationic compound SuperFect reporter vector complexes were transfected into 293T cells,The dual-luciferase Report Assay System was used to quantitate the reporter vector luciferase activity and located the core promoter position.The SIRT1 recombinants were transfected into AML1-ETO positive and negative cell lines, and transfected into 293T cells with different doses of AML1-ETO plasmids,using dual-luciferase Reporter Assay System to further research on the regulation of AML1-ETO on SIRT1 core promoter transcription activity.In the second part of the study,quantitative RT-PCR and Western blot were used to observe the change of mRNA and protein expression levels of EZH1 in AML1-ETO positive and negative cell lines.EZH1 core promoter sequences were amplified by PCR from the transciptional start site to build differnet luciferase reporter vetors,then they were transfected into AML1-ETO positive and negative cell lines after wrapped with SuperFect,at last,measured luciferase activity by dual-luciferase Report Assay System to locate core promoter of EZH1.After revealing that there were many AML1 binding sites in the promoter region of EZH1 by biological information,we built mutant plasmids to transfect with corresponding non-mutant plasmids into AML1-ETO positive and negative cell lines in order to explore the binding of AML1-ETO in EZH1 promoter region and ChIP was used for confirmation.At last using CCK-8, PI staining method to analyze EZH1 influence on cell proliferation and cell cycle.Results:The first part,RT-PCR revealed the higher expression levels of SIRT1 mRNA in AML1-ETO positive clinical samples of leukemia patients compared with the normal donors and also in AML1-ETO positive cell lines (U937AE and SKNO-1) compared with negative cell lines.Six kinds of SIRT1 plasmids were constructed and used Dual-luciferase reporter system to detect their luciferase activity and deduced that the location of the core promoter is between 1769bp to 1060bp.The SIRT1 recombinants were transfected into AML1-ETO positive and negative cell lines and the luciferase activity results showed that core promoter activity was significantly reduced in cell lines knockout AML1-ETO;and in 293Tcells with different dose of AML1-ETO plasmids,the luciferase activity results showed that the expression level of SIRT1 increased with the increasement of AML1-ETO dose.The second part,quantitative RT-PCR and Western blot revealed the higher expression levels of EZH1 mRNA and protein in AML1-ETO positive cell lines (U937AE and SKNO-1).We constructed 8 kinds of EZH1 recombinants and 4 kinds of mutant recombinants and located the core promoter region of EZH1(1126-1164bp)by measuring the relative luciferase activity of the 8 recombinants,then assured the binding site of AML1-ETO in EZH1(+1109bp) by transfected mutant recombinants together with corresponding non-mutant recombinants into AML1-ETO positive and negative cell lines.ChIP showed the results that AML1-ETO could bind to the promoter of EZH1 in core promoter region.EZH1 silent expression can cause inhibition of proliferation and cell cycle in AML1-ETO positive leukemia cell.Conclusion:The first part:SIRT1 may be one of the target genes of AML1-ETO.The expression level of SIRT1 is positively correlated with AML1-ETO,suggesting that AML1-ETO can promote the activity of SIRT1 promoter to up-regulate its transcription.This study provide experimental basis for in-depth study of highly expressed SIRT1 gene in leukemia and targeting leukemia stem cells by selective inhibition of SIRT1.The second part:Histone methylation is an important mechanism of t(8;21) AML, AML1-ETO can combine with EZH1 promoter region to make aberrant transcription of EZH1,and change transcriptional status of other genes by histone methylation,which results in activation of cell proliferation and cell cycle and epigenetics alterations involved with the development of leukemia.The study finds out the bind site of AML1-ETO on EZH1 core promoter,it provides a theoretical basis for the new clinical treatments.