A Preliminary Study On Super Enhancer Related Oncogenes In Children With Acute Myeloid Leukemia

Part Ⅰ:Screening of Super Enhancer Spectrum associated with Acute Myeloid Leukemia in ChildrenObjective:The incidence of childhood acute myeloid leukemia(AML)accounts for about 20%of childhood leukemia,but the mortality rate accounts for more than 50%.At present,children with 10%～30%AML are ineffective to the first-line standard induction regimen.40%to 80%of the children will eventually relapse even if they are in complete remission,and more than 50%of the recurrent children will die due to treatment failure,so AML is a difficult point in the treatment of hematological tumors in children.At present,it is believed that the reason of refractory and easy recurrence is the lack of understanding of its pathogenesis and the lack of specific targeted drugs.In this paper,by constructing the leukemia super enhancer map,we want to find the super enhancer oncogenes associated with childhood acute myeloid leukemia and obtain a new candidate strategy for the treatment of AML.Methods:Collected bone marrow samples from 11 newly diagnosed children with acute myeloid leukemia,and performed Chromatin Immunoprecipition(ChIP)and highthroughput sequencing for histone H3 Lysine 27 acetylation(H3K27ac)and high-throughput sequencing,using bioinformatics Methods Analyze and obtain super-enhancers,compare the data of normal human bone marrow and other tumor cell lines in the NCBI database,and find the mRNA expression of related genes in the CCLE database.Use this as a reference to analyze the super-enhancement related to AML in children The clinical significance of the child.Results:The super enhancer maps of bone marrow samples from 11 newly diagnosed children with AML were constructed by chromatin immunoprecipitation sequencing(ChIPseq),and the peak value of 15000～81000peaks H3K27ac was obtained.the number of super enhancers in a single sample ranged from 150 to 1800.Among them,super enhancers have been reported in other tumors:GFI1,ZFP36L2 genes,but also found many unknown super enhancer regulatory genes,such as ZEB2/ZEB2-AS1,IRF5,CEBPG.The CCLE database indicated that ZEB2/ZEB2-AS1,IRF5,and CEBPG have higher mRNA expression levels in AML than other tumors.Conclusion:Screening and discovering the super-enhancer spectrum of AML in children,and obtaining a series of super-enhancer regulation genes related to AML in children,such as ZEB2,IRF5 and CEBPG,etc.Part Ⅱ:A preliminary study on the functional mechanism of CCAAT enhancer binding protein gamma(CEBPG)in acute myeloid leukemia cell lines.Objective:In the early stage,we analyzed the super enhancer map of 11 newly diagnosed children’s AML bone marrow samples by ChIP-seq high-throughput sequencing and bioinformatics,which showed that there were super-enhancer elements near CEBPG.It is speculated that CEBPG may be regulated by super-enhancer.So far,the function and mechanism of CEBPG gene in childhood acute myeloid leukemia are not clear,so its function and mechanism have been studied.Methods:The gene interference system was used to down-regulate the expression of CEBPG gene in acute myeloid leukemia cell lines,and the interference efficiency on CEBPG was detected by Western Blot and RT-qPCR.The functional experiments of these cells were carried out.The inhibitory effect of knockdown on the growth of acute myeloid leukemia cell lines was evaluated by CCK-8.Western Blot was used to detect PARP cleavage,Annexin V/PI staining and flow cytometry were used to determine the effect of knock-down on apoptosis of acute myeloid leukemia cells.The downstream genes and pathway hints related to the CEBPG gene were obtained by RNA-seq analysis of the AML cell line which knocked down the CEBPG.Once again,the gene interference system was used to knock down the expression of downstream genes to verify its function.Results:Western Blot detection showed that CEBPG protein was highly expressed in myeloid leukemia cells.The results of CCK-8 detection showed that the proliferation ability of cells in CEBPG gene knockout group decreased significantly(P<0.001,with statistical difference).Knockdown CEBPG cells showed enhanced PARP cleavage and higher apoptosis rate(P<0.05,with statistical difference).RNA-seq analysis showed that eukaryotic translation initiation factor 4e binding protein 1(EIF4EBP1)was significantly down-regulated in AML cell lines(NB4 and MV4-11).The study on the function of EIF4EBP1 showed that the proliferation ability of AML cells decreased after EIF4EBP1 gene knockdown by CCK-8(P<0.0001,with statistical difference).Western Blot detection showed that PARP cleavage was enhanced,and the proportion of apoptosis was significantly increased by Annexin-V flow cytometry(P<0.001,with statistical difference).Conclusion:Super enhancer regulatory gene CEBPG plays a key role in AML,and interfering with its expression can promote apoptosis and proliferation inhibition of myeloid leukemia cells.EIF4EBP1 is probably the key downstream gene in which CEBPG plays a role.