The Expression Of EZH2and Its Relationship With DNA Methyltransferases Genes In Patients With Nomal-cytogenetics Acute Myeloid Leukemia

Objective: Acute myeloid leukemia (AML) is a heterogeneoushematologic malignant tumors. And cytogenetics is one of the importantfactors that effects on treatment and prognosis. Patients with CN-AML, whichapproximately account for forty-five percent of all AML patients, isgenetically heterogeneous. Research on gene mutation in CN-AML patientscontributes to distinguish genetic injury associated with teatment failure.Laboratory studies have implicated epigenetic dysregulation as acommon pathogenetic mechanism in acute myeloid leukemia (AML). Theessential epigenetic systems involved in repression of gene activity are thePolycomb group (PcG) proteins and the DNA methylation system. Theabnormal alterations of these systems, which lead to the hypersilence of tumorsuppressor, play potential roles in tumorigenesis.The EZH2(enhancer of zeste homolog2, EZH2) gene is a homolog ofthe Drosophila PcG gene enhancer of zest. It is mapped at chromosome7q35and frequently over-expressed in a wide variety of cancerous tissue types. As aPcG protein, EZH2is the catalytic subunit of Polycomb repressive complex2(PRC2), which is the major enzyme that methylates lysine27of histone H3(H3K27). Through the enzymatic modification of chromatin, histonemethylation mediated by PRC2plays an important role of chromatin silencing.And this silencing effect targets on a set of genes involved in cell-cycleregulation and differentiation. Although the tumorigenesis mechanismassociated with EZH2has not been completely revealed, there is ampleevidence that the PcG silencing system contributes to oncogenesis.Another well-characterized epigenetic alteration is CpG DNAhypermethylation which often accumulates in promoter regions of tumor suppressor genes, thereby contributing to tumor suppressor loss throughepigenetic silencing. The DNA methylation is mediated by DNAmethyltransferases (DNMTs). Notably, genome-wide methylation studies haveidentified a set of genes recurrently targeted by aberrant promoterhypermethylation in AML. And the over-expression of DNA methyltransferasegenes, which include DNMT1, DNMT3A and DNMT3B, have been identifiedin patients with AML in previous studies.PcG silencing and DNA methylation were initially consideredbiochemically independent gene silencing systems. However, the latter studiesin human cells shows that EZH2and DNA methyltransferases are physicallyand functionally linked, thus uncovering a previously unrecognized directconnection between two key epigenetic repression systems. By determiningthe expression of EZH2DNMT1DNMT3A and DNMT3B in patients withCN-AML, the study is to analyse the relationship between EZH2and DNMTs,and explore the possible pathogenesis and prognostic effect associated withEZH2in CN-AML.Methods: The expression level of EZH2DNMT1DNMT3A andDNMT3B mRNA were determined in78patients with AML (including27denovo acute myeloid leukemia patients,8relapsed patients (RP),43completeremission patients (CR),all de novo and RP patients are CN-AML), and11samples of normal controls (NC) by semi-quantity reverse transcription-polymerase chain reaction (RT-PCR).Results:1The expression level of EZH2DNMT1DNMT3A andDNMT3B mRNA in CN-AML patients1.1The expression level of EZH2mRNA in de novo group is0.151(0.465) and positive rate is96.3%(26/27), in RP group is0.511(0.451) and100%(8/8), CR group is0(0.177) and48.8%(21/43) and NC group is0.018(0.202) and54.5%(6/11). The expression level of EZH2mRNA in de novoand RP group are both significantly higher than in CR and NC group (P <0.05). The positive rate in de novo and RP group are also both significantlyhigher than NC group (P <0.05). There are no significantly differences between any other tow groups (P>0.05).1.2The expression level of DNMT1mRNA in de novo group is0.499(0.31) and positive rate is96.3%(26/27), in RP group is0.502(0.581) and87.5%(7/8), CR group is0.161(0.393) and69.8%(30/43) and NC group is0.203(0.293) and72.7%(8/11). The expression level of DNMT1mRNA in denovo and RP group are both significantly higher than in CR and NC group (P<0.05). There are no significantly differences between any other tow groups(P>0.05).1.3The expression level of DNMT3A mRNA in de novo group is0.722(0.934) and positive rate is100%(27/27), in RP group is0.733(0.673)and100%(8/8), CR group is0.226(0.461) and81.4%(35/43) and NC group is0.134(0.472) and90.9%(10/11). The expression level of DNMT3A mRNA inde novo and RP group are both significantly higher than in CR and NC group(P <0.05). There are no significantly differences between any other towgroups (P>0.05).1.4The expression level of DNMT3B mRNA in de novo group is0.071(0.387) and positive rate is92.6%(25/27),in RP group is0.18(0.536) and87.5%(7/8),CR group is0(0.021) and39.5%(17/43) and NC group is0(0.092) and45.5%(5/11). The expression level of DNMT3B mRNA in denovo and RP group are both significantly higher than in CR and NC group (P<0.05). The positive rate in de novo group is also significantly higher thanNC group (P <0.05). There are no significantly differences between any othertow groups (P>0.05).2According to the tracking examination of16patients, the expressionlevel of EZH2in untreated AML patients significantly decrease from0.151(0.72) to0.033(0.167), DNMT1from0.709(0.538) to0.317(0.475),DNMT3A from0.798±0.606to0.347±0.284, and DNMT3B from0.051(0.884) to0(0.165), when the patients get CR (P <0.05).3According to median expression level of EZH2, de novo group isdivided into high-expression group and low-expression group. The CR rate inhigh-expression group is71.4%, and in low-expression group is76.9%, there is no significantly difference between them, as well as death rate (P>0.05).4In de novo AML patients, there is no correlation between theexpression level of EZH2mRNA and the quantity of Peripheral WBC andbone marrow cells which expressed CD34(P>0.05).5Positive correlation in de novo group is not only revealed among allthree DNMT gens, but also between EZH2and DNMTs (rE-D1=0.403; rE-3A=0.546; rE-3B=0.453; rD1-3A=0.439; rD1-3B=0.401; r3A-3B=0.576. P<0.05).Conclusions:1Compared with NC group, the expression level andpositive rate of EZH2mRNA in de novo and RP group are significantly higher.And the level significantly reduces after paitients get CR. Histone methylationassociated with EZH2leads to chromatin silence. So we consider that EZH2may play an important role in the pathogenesis of CN-AML.2According to median expression of EZH2mRNA, the de novo group isdivided into high-expression and low-expression group, and there is nosignificantly difference between the CR rate of these tow groups, as well asdeath rate. Therefore we think that in CN-AML, EZH2may not be an indexthat predicts poor treatment effectiveness and unfavorable prognostic as inepithelial alignancies.3Expression of all three DNMT genes in de novo and RP group aresignificantly higher than that in NC group. And Positive correlation in de novogroup is not only revealed among all three DNMT genes, but also betweenEZH2and DNMTs. So we consider that DNA methylation plays an importantrole in the pathogenesis of CN-AML, and coexpression between EZH2andDNMTs demonstrates that histone modification associated with EZH2andDNA methylation are functionally linked in CN-AML. Our result cansomehow provide theoretical basis to new treatment Protocols.