AML1对ZFP36L2的转录调控及ZFP36L2对急性髓系白血病细胞生物学功能的影响

发布时间：2018-08-24 08:08

【摘要】：研究目的:染色体t(8;21)(q22;q22)异位形成AML1-ETO融合基因,AML1-ETO融合蛋白能异常募集组蛋白脱乙酰化酶(Histone Deacetylase,HDAC)进而抑制AML1靶基因的正常转录。HDAC抑制剂(HDACi)苯丁酸钠(phenylbutyrate,PB)在处理AML1-ETO+ Kasumi-1细胞时能够诱导凋亡分化,并伴随AML1、PIG7等多种基因表达上调。通过生物信息学分析发现,表达上调的ZFP36L2基因启动子区有AML1的潜在结合位点。ZFP36L2是含CCCH锌指结构的RNA结合蛋白,通过加快mRNA的降解或抑制蛋白翻译从而调控特异mRNA的表达。本研究旨在探讨AML1对ZFP36L2是否具有转录调控作用,以及ZFP36L2蛋白在AML细胞系Kasumi-1和HL-60中的生物学功能,并探讨其作用机制。研究方法:实时定量PCR(qRT-PCR)检测ZFP36L2在AML1-ETO+患者细胞和细胞系中的表达。应用qRT-PCR和Western blot技术,检测PB处理Kasumi-1细胞后ZFP36L2mRNA和蛋白表达变化。构建包含AML1结合位点的ZFP36L2启动子报告质粒,应用双荧光素酶报告基因系统,检测转录因子AML1、AML1-ETO对ZFP36L2的转录调控作用,并在CV-1细胞中分别转入外源AML1和AML1-ETO,应用qRT-PCR和Western blot检测内源ZFP36L2 mRNA和蛋白水平变化。利用四环素可诱导表达体系Tet-On系统,构建慢病毒表达载体pLVX-Tight-Puro-ZFP36L2,通过PEI转染方法,转染HEK293T细胞,收获病毒,对Kasumi-1细胞和HL-60细胞进行感染,筛选稳定克隆。在强力霉素(doxycycline,Dox)诱导时,外源ZFP36L2基因可以表达,而未经Dox诱导,外源导入的ZFP36L2基因不表达,即可以通过Dox来控制该基因的表达与否。应用qRT-PCR和Western blot,检测ZFP36L2在mRNA和蛋白水平的可诱导表达。诱导ZFP36L2过表达时,检测Kasumi-1和HL-60细胞的增殖、凋亡、周期和分化等细胞生物学功能的改变,通过Western blot检测凋亡蛋白和周期蛋白的改变。研究结果:ZFP36L2在白血病患者中低表达,且ZFP36L2在M2b患者中的表达水平低于在非M2b患者中中表达水平,在AML1-ETO+细胞系Kasumi-1细胞中表达水平相对较低。PB处理诱导Kasumi-1细胞凋亡时,qRT-PCR和Western blot检测显示ZFP36L2在mRNA和蛋白水平表达均上升。AML1激活ZFP36L2启动子报告质粒,且激活作用呈剂量依赖性,而AML1-ETO对ZFP36L2启动子报告质粒的转录激活没有明显作用。在CV-1细胞中过表达AML1可以上调内源ZFP36L2的蛋白表达;而过表达AML1-ETO对ZFP36L2的蛋白表达无明显影响。当ZFP36L2基因在Kasumi-1细胞和HL-60细胞中诱导表达时,细胞均表现增殖能力受抑制,细胞周期阻滞在G0/G1期,细胞凋亡增加,未检测到细胞分化。相关凋亡蛋白和周期蛋白,出现相应改变。研究结论:本研究发现了 AML1新的靶基因—ZFP36L2。AML1转录调控激活ZFP36L2表达,过表达ZFP36L2抑制细胞增殖,阻滞细胞周期,诱导细胞凋亡,是肿瘤抑制因子。当发生t(8;21)染色体异位形成AML1-ETO融合蛋白,上述作用减弱或消失,促进白血病发生。本研究阐明了 AML1对ZFP36L2的调控作用,以及ZFP36L2在白血病细胞中的生物学作用。
[Abstract]:Objective: to investigate the heterotopic formation of AML1-ETO fusion gene (AML1-Eto fusion protein) on chromosome t _ (8) (q22) and to recruit histone deacetylase (Histone Deacetylase,HDAC) to inhibit the normal transcription of AML1 target gene. HDAC inhibitor, (HDACi) phenylbutyrate (phenylbutyrate,PB), in the treatment of AML1-ETO Kasumi-1 cells Can induce apoptosis and differentiation, The expression of many genes such as AML1,PIG7 was up-regulated. Bioinformatics analysis showed that the up-regulated ZFP36L2 gene promoter had the potential binding site of AML1. ZFP36L2 was a RNA binding protein containing CCCH zinc finger structure, which regulated the expression of specific mRNA by accelerating the degradation of mRNA or inhibiting the translation of mRNA. The aim of this study was to investigate the transcriptional regulation of AML1 on ZFP36L2 and the biological function of ZFP36L2 protein in AML cell line Kasumi-1 and HL-60, and to explore its mechanism. Methods: real-time quantitative PCR (qRT-PCR) was used to detect the expression of ZFP36L2 in AML1-ETO cells and cell lines. QRT-PCR and Western blot techniques were used to detect the changes of ZFP36L2mRNA and protein expression in Kasumi-1 cells treated with PB. The reporter plasmid of ZFP36L2 promoter containing AML1 binding site was constructed, and the transcriptional regulation of ZFP36L2 by the transcription factor AML1,AML1-ETO was detected by using double luciferase reporter gene system. Exogenous AML1 and AML1-ETO, were transferred into CV-1 cells respectively to detect the changes of endogenous ZFP36L2 mRNA and protein levels by qRT-PCR and Western blot. The lentivirus expression vector pLVX-Tight-Puro-ZFP36L2, was constructed by using tetracycline induced expression system Tet-On system. The lentivirus expression vector pLVX-Tight-Puro-ZFP36L2, was transfected into HEK293T cells by PEI transfection, and the virus was harvested. Kasumi-1 cells and HL-60 cells were infected and stable clones were screened. During the induction of doxycycline (doxycycline,Dox), the exogenous ZFP36L2 gene can be expressed, but without the Dox induction, the exogenous ZFP36L2 gene can not be expressed, that is, the expression of the gene can be controlled by Dox. QRT-PCR and Western blot, were used to detect the inducible expression of ZFP36L2 at mRNA and protein levels. When ZFP36L2 overexpression was induced, the proliferation, apoptosis, cell cycle and differentiation of Kasumi-1 and HL-60 cells were detected. The changes of apoptotic protein and cyclin were detected by Western blot. The results showed that the expression level of ZFP36L2 in M2b patients was lower than that in non-M2b patients, and the expression of ZFP36L2 in leukemia patients was lower than that in non-M2b patients. The expression level of ZFP36L2 in Kasumi-1 cells was relatively low. PB treatment induced apoptosis of Kasumi-1 cells. The results of qRT-PCR and Western blot showed that the expression of ZFP36L2 increased at mRNA and protein levels. AML1 activated ZFP36L2 promoter reporter plasmid in a dose-dependent manner. However, AML1-ETO had no significant effect on the transcriptional activation of ZFP36L2 reporter plasmids. Overexpression of AML1 could up-regulate the protein expression of endogenous ZFP36L2 in CV-1 cells, but overexpression of AML1-ETO had no effect on the expression of ZFP36L2 protein. When ZFP36L2 gene was induced to express in Kasumi-1 cells and HL-60 cells, cell proliferation was inhibited, cell cycle was blocked in G0/G1 phase, apoptosis was increased, and cell differentiation was not detected. The related apoptotic proteins and cyclins changed accordingly. Conclusion: the new target gene of AML1-ZFP36L2.AML1 transcriptional regulation activates the expression of ZFP36L2. Overexpression of ZFP36L2 inhibits cell proliferation, blocks cell cycle, induces apoptosis, and is a tumor suppressor. When the t (8) chromosome ectopic formed AML1-ETO fusion protein, the above effects were weakened or disappeared, and promoted the development of leukemia. The purpose of this study was to elucidate the regulatory effect of AML1 on ZFP36L2 and the biological role of ZFP36L2 in leukemia cells.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71

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