EZH2 High Expression In AML Patients And The Anti-Tumor Effect Of Its Inhibitor DZNep With Its Molecualr Mechanism In AML

Background and Objective: EZH2(enhancer of zeste homolog 2)is a core member of polycomb repressive complex 2(PRC2)with histone methyltransferase activity,which can catalyze the methylation of histone 3 lysine 27.This set of histone modification mediated epigenetic silencing of genes is one of the ways in which many tumor suppressor genes are inactivated.In addition,EZH2 is overexpressed in various cancers such as breast cancer,prostate cancer and colon cancer,suggesting that EZH2 has the role of promoting the development of cancer,so it is regarded as one of the targets of cancer treatment.DZNep is the first EZH2 small molecule inhibitor reported by drug screening.Many studies have applied it to cancer patients with abnormally elevated EZH2,and have achieved anti-tumor effects.However,it is poorly understand the effect of DZNep in acute myeloid leukemia(AML).Therefore,this study aims to examine the EZH2 expression in AML and analyze the association of EZH2 expression with the prognosis,observe the effect of DZNep on the proliferation,apoptosis and cell cycle progress in the U937 AML cells and also examine the underlying mechanism of DZNep anti-tumor effect.This study is mainly divided into three parts:(1)the EZH2 expression in adult AML patients and its clinical significance;(2)the vitro effect of EZH2 inhibitor DZNep in AML cells;(3)The mechanism underlying anti-tumor effect of EZH2 inhibitor DZNep by identification of the potential key genes and signaling pathways through high-throughput transcriptome sequencing and bioinformatics analysis.Methods: RT-qPCR(real-time quantitative polymerase chain reaction)was used to detect the expression of EZH2 mRNA in primary cells from AML patients.Cell proliferation of U937 upon EZH2 inhibitor DZNep treatmentwas measured by CCK-8(Cell Counting Kit-8)assay,cell apoptosis by Annexin V PE / 7-AAD double staining following flow cytometry analysis and cell cycle by PI single staining.RNA-Seq was used to identify mRNA expression profiling and differentially expressed genes(DEGs)in the DZNep-treated cells versus non-treatment control.Expression of DEGs was verified by RT-qPCR assay.CBio Portal,AMC humancancer and GEPIA databases were used to analyze the the key genes’ function and pathway of DEGs.Results: The expression of EZH2 mRNA is significantly higher in newly diagnosed adult AML patients than that in the normal control;patients with EZH2 high expression is associated with shorter survival versus that of low expression.EZH2 inhibitor DZNep inhibits U937 cell proliferation,induces cell apoptosis and arrests cell cycle.We identified the global gene expression profiling upon DZNep treatment in U937 cells by RNA-seq,and analyzed DEGs with DESeq software,2761 DEGs were identified with the criteria of | log2 Foldchang |> 1,P value <0.025,of which 1362 were up-regulated and 1399 were down-regulated.The GO(Gene Ontology)analysis showed that DEGs are related to tumor-related biological processes,cell component and molecular functions;KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis revealed that DEGs were involved in 94 signal pathways.Through c Bio Portal,AMC humancancer and GEPIA database,we also identified the key genes(SSH3,MCM3-7,TNF,REM2,PIK3IP1,VWA5 A and SPRY4),and important pathways incvolving in cell cycle progress,regulation of apoptosis,metabolism and multi-oncogenic signaling(MAPK,m TOR,AMPK,Wnt,Notch etc).We also found that the aberrent expression of these key genes in AML patients through Metadata analysis of microarray and RNA-seq data,and their aberrent expression is related to the poor survival of AML patients,revealing that these genes play an important role in the anti-tumor effect mediated by DZNep.Conclusion: EZH2 expression is significantly increased in AML,and the EZH2 high expression is associated with poor outcome.EZH2 inhibitor DZNep has anti-tumor effect in AML cells by inhibition of the cell proliferation,induction of apoptosis and arrestment of cell cycle.DZNep anti-tumor effect in AML is mediated by regulation of key genes and signaling pathways.This study further indicates EZH2 plays important roles in oncognesis and development of AML,and also lies an experimental foundation for further research on the efficacy of DZNep in AML.Our research also identified several new oncogenes and tumor suppressor genes in AML,and we will extensively study their functions in the future.