AML1-Evi1基因致急性髓系白血病的分子机制研究（斑马鱼模型）

Acute myeloid leukemia is related to abnormal chromosome and genetic mutations. The AML1-Evi1 fusion gene is seen more at in acute myeloid leukemia and the prognosis is poor. Although AML1 and Evi1, as a function of a single gene, have been reported in the literature, how dose the fusion gene make the hematopoietic stem cells and progenitor cells with poor differentiation or hindered mechanism has been still unclear. Effect of differences between AML1-Evi1 and single gene of AML1 and Evi1 is not clear. The subject make use of the unique advantages of zebrafish with development and disease model organism, we have established stable transgenic line of Tg(AML1-Evi1:EGFP) zebrafish model in the early stage based on the research work, and reestablished stable transgenic line of Tg (AML1:EGFP) and Tg (Evi1:EGFP) zebrafish model. Through transcriptome sequencing, whole-mount situ hybridization, RNA interference technology in vivo and other technologies, further study of the function of AML1-Evi1 and illustrate the AML1-Evi1 fusion gene induced uniquely the leukemia mechanism and pathway. Ultimately, basis on the above research, high throughout drug screening will be carried out on this AML1-Evi1 zebrafish model in vivo. Guiding drug candidate will be identified for specific target genes of AML1-Evi1. Finally, the candidate drugs will be proved in the umbilical cord blood CD34+ cells transfected with AML1-Evi1 plasmid and patients' leukemia cells in vitro, in order to clarify inhibition of AML1-Evi1 target gene treatment feasibility in AML. The applicant has rich experience in leukemia drug resistance research and transgenic zebrafish technology It is expected that the result of this research can provide a new method for targeted therapy and have groundbreaking significance.

急性髓系白血病发生与染色体及基因异常有关，AML1-Evi1融合基因多见急性髓细胞白血病且预后极差。该基因使造血干/祖细胞分化不良或受阻的机制及其与单基因的作用差异仍未明确。本课题利用斑马鱼发育与疾病模式生物体的独特优势，在前期已建立Tg(AML1-Evi1:EGFP)斑马鱼研究工作基础上，再建Tg(AML1:EGFP)和Tg(Evi1:EGFP)斑马鱼模型，通过转录组测序，原位杂交，活体RNA干扰等技术进行功能比对，深入研究AML1-Evi1的功能及AML1-Evi1融合基因独特的致白血病通路和靶基因，并进行活体高通量药物筛选，识别特异性针对AML1-Evi1靶基因的先导候选药物。最后在转染AML1-Evi1质粒的脐带血CD34+细胞、AML1-Evi1阳性患者原代细胞中进行体外验证，以明确抑制AML1-Evi1白血病靶基因治疗在AML中的可行性，为靶向治疗提供新的思路，具有开创性意义。

摘要：本课题利用斑马鱼模式生物体的独特优势，在前期已建立Tg(AML1-Evi1:EGFP)斑马鱼研究工作基础上，又再建Tg（TOL2-Evi1：EGFP）（TE）和Tg( TOL2-AML1:EGFP)（TA）斑马鱼模型。通过转录组测序，分子基因检测，原位杂交等技术进行功能比对，深入研究AML1-Evi1的功能及AML1-Evi1融合基因独特的致白血病通路和靶基因，并进行活体高通量药物筛选，识别特异性针对AML1-Evi1靶基因的先导候选药物三氧化二砷（ATO）。并在Evi1高表达的急性髓细胞白血病细胞株THP-1 和Evi1阳性患者原代细胞中进行作用机制研究，结果发现：（1）提示转基因型斑马鱼造血细胞增殖能力较野生型增高，相较于正常野生型斑马鱼，Evi1过度表达的斑马鱼出现明显的早期发育延迟和后期的多器官畸形，并能够促进髓系细胞的永生化，Tg（ TOL2-Evi1:EGFP）（TE）斑马鱼对比野生型斑马鱼其存活率明显降低。（2）利用基因芯片，对WT和Tg(AML1-Evi1:EGFP)斑马鱼胚胎进行基因分析，发现AML1-Evi1可以下调Jnk信号通路上一些关键的基因，包括jun, atf2, elk1和 ddit3。蛋白电泳显示在Tg(AML1-Evi1:EGFP)斑马鱼中Jnk 和p-Jnk下降。（3）EVI-1转基因斑马鱼外周血细胞全基因组RNA-Seq，发现在MAPK、细胞周期调节、局灶性粘附和血管内皮生长因子信号传导中，受EVI-1过表达功能簇影响最大的基因。蛋白电泳进一步发现TE的F2代中p44/42MAPK(ERK1/2)蛋白表达显著增加。（4）ATO能明显降低TE斑马鱼中Evi1基因的表达及相关造血因子的mRNA水平。（5）EVI-1表达阳性的AML患者原代细胞，经测序检测，表达全长序列的EVI-1，并存在3处突变位点，ATO能下调该患者原代白血病细胞中EVI-1 mRNA的表达。（6）体外高表达Evi1基因的THP-1细胞株研究发现，ATO通过下调Evi1 mRNA和EVI1蛋白，减弱EVI-1对JNK信号通路的抑制，从而激活JNK信号通路，继而激活Bcl-2家族和线粒体通路，诱导THP-1细胞株凋亡。