The Transcriptional Regulatory Relationship Between AML1 And ZFP36L2 And The Effects Of ZFP36L2 On The Biological Functions Of AML Cell Lines

Objective:The t(8;21)(q22;q22)translocation forms the fusion gene AML1-ETO,which contains the Runt homology domain of AML1 and nearly the whole ETO gene.The fusion protein AML1-ETO recruits an inhibitive complex,consisting of nuclear receptor co-repressor/mSin3/histone deacetylase(HDAC),which mediates transcriptional repression of AML1 target genes.Sodium phenylbutyrate(PB),HD AC competitive inhibitor,can induce apoptosis of Kasumi-1,accompanying upregulation of many genes,including AML1 and PIG7,etc.Among those genes,we suppose some genes are potential targets of AML1.Based on the bioinformatics analysis,there are AML1 binding sites on the promoter of ZFP36L2 gene which is upregulated by PB treatment.ZFP36L2 is RNA-binding protein and interacts with AU-rich elements in the 3’UTR of mRNA,leading to mRNA degration and translational repression.The purpose of this study is to explore the transcriptional regulatory relationship between AML1 and ZFP36L2,and to detect the biological function of ZFP36L2 on AML cell lines and related mechanisms.Methods:Real-time quantitative PCR(qRT-PCR)was used to detect the ZFP36L2 expression in M2b patients’ cell and AML1-ETO+ cell lines.And qRT-PCR and Western blot assay were performed to examine the expression of ZFP36L2 in Kasumi-1 cells after PB treatment.The pGL3-ZFP36L2-promoter reporter plasmid with AML1 binding site was constructed.The luciferase report assays were performed to investigate the roles of AML1 and AML1-ETO on transactivation of ZFP36L2.The exogenous AML1 and AML1-ETO were transfected into the CV-1 cells,and the endogenous ZFP36L2 expression was measured by qRT-PCR and Western blot.The tetracycline(Tet)inducible Tet-on system was used to construct the ZFP36L2 expression plasmid pLVX-Tight-Puro-ZFP36L2.And lentivirus was packaged to infect the Kasumi-1 and HL-60 cell lines.Kasumi-1 and HL-60 with ZFP36L2 stably expressed were screened by flow cytometer and puromycin.The expression of ZFP36L2 was turned on when selected cells were exposed to doxycycline(Dox)in the tet-on inducible system.The qTR-PCR and Western blot were used to detect the inducible expression of ZFP36L2.The biological functions of ZFP36L2 by MTS experiments and flow cyctometer were assayed.The associated cell cycle and apoptotic protein were evaluated by Western blot.Results:It showed ZFP36L2 was lower expressed in M2b patients and AML1-ETO+ cell lines.And the mRNA and protein level of ZFP36L2 were upregulated in Kasumi-1 cells line treated with PB.AML1 transactivated the expression of pGL3-ZFP36L2-promoter reporter plasmid in a dose-dependent manner,while no significant upregulations were observed when co-transfected with AML1-ETO.Besides,the transient expression of exogenous AML 1 promoted the endogenous expression of ZFP36L2 in CV-1 cells.Overexpression of ZFP36L2 in Kasumi-1 and HL-60 cells could inhibite cell proliferation,promote cell-cycle arrest in G0/G1 phase and induce apoptosis.Western blot analysis showed down-regulation of anti-apoptotic proteins and up-regulation of pro-apoptotic protein,while the expression of Cyclin-CDK complex,requird for the entry in the S phase of cell cycle,was reduced.Conclusions:In this study,ZFP36L2 was found to be transactivated by AML1.And ZFP36L2 could inhibite cell proliferation,block cell cycle and induce apoptosis in acute leukemia cell lines.