t(8;21)急性髓系白血病中选择性剪切体AML1-ETO9a产生的表观遗传学机制研究

t(8;21) is one of the most frequent chromosomal translocations occurring in acute myeloid leukemia (AML), and the fusion gene AML1-ETO is considered as a significant characteristics and leukemia-initiating event in t(8;21) AML. A lots of researches confirmed that alternative splicing variants AML1-ETO9a are closely related with occurrence, development and prognosis of t(8;21) AML. However, it is not clear what the role that epigenetic regulation mechanisms played in producing of alternative splicing variants AML1-ETO9a. Our preliminary results shown that that splicing factors SRSF2 and SRSF6 promote alternative splicing of AML1-ETO9a and significantly enhanced cell malignant proliferation, especially, this effects are cell specific. Bioinformatics analysis and preliminary experiments speculated that fusion gene AML1-ETO can bind the promoter regions and recruit epigenetic regulators and raised the expression of splicing factors SRSF2 and SRSF6, promote the producing of alternative splicing variants AML1-ETO9a in transcriptional level; On the other hand, the fusion protein AML1/ETO make miR-9-1 epigenetic silenced by recruiting epigenetic enzymes in promoter region of miR-9-1, in which silenced miR-9-1 released the inhibition of AML1-ETO9a in transformation level. The increased expression level of AML1-ETO9a play a role as transcriptional factor and bind and recruit epigenetic enzymes on the promoter of downstream target gene c-KIT and epigenetic increase the expression level of c-KIT, then activate PI3K signal pathway, lead to t(8;21) leukemogenesis. In addition, preliminary study of the occurrence of AML1-ETO9a, together with elucidation of the epigenetic regulatory mechanisms in t(8;21) AML, laid a foundation for the mechanism of the occurrence and recurrence of AML, also provides a new angle of view for us to fully understand the complex gene regulation network, in the end of clinical diagnosis of leukemia and other cancers, which can be successfully targeted by pharmacological treatment using de-epigenetic agents in t(8;21)-leukemias.

融合基因AML1-ETO是t(8;21)急性髓系白血病的显著特征和始发因素。研究证实，选择性剪切本AML1-ETO9a与此类白血病的发生、发展及预后密切相关，但表观遗传学调控机制在AML1-ETO9a产生中的作用目前尚不清楚。前期结果表明：剪切因子SRSF2/6促进了AML1-ETO9a的选择性剪切，显著增强了细胞的恶性增殖，且此作用具有细胞特异性。生物信息学分析推测：AML1-ETO通过募集乙酰转移酶使剪切因子SRSF2/6发生表观遗传学上调，促进选择性剪切体的产生；另一方面，通过募集表观遗传学修饰酶使miR-9-1发生表观遗传学沉默，解除了对AML1-ETO9a翻译水平上的抑制，促使其结合到重要的下游靶基因c-KIT的启动子区，并促进其异常表达，激活PI3K等信号途径，导致白血病的发生。这为阐明t(8;21)AML的发生和复发奠定了基础，也为白血病的诊断、治疗和预后提供新的手段和策略。

融合基因AML1-ETO是t(8;21)急性髓系白血病的显著特征和始发因素。研究证实，选择性剪切本AML1-ETO9a与此类白血病的发生、发展及预后密切相关，但表观遗传学调控机制在AML1-ETO9a产生中的作用目前尚不清楚。本项目通过深入研究证实：1）剪切因子SRSF2/6促进了AML1-ETO9a的选择性剪切，显著增强了细胞的恶性增殖，且此作用具有细胞特异性。2)生物信息学分析推测：AML1-ETO通过募集乙酰转移酶使剪切因子SRSF2/6发生表观遗传学上调，促进选择性剪切体的产生；3)另一方面，通过融合蛋白AML1-ETO可以募集表观遗传学修饰酶，如DNMT3A、DNMT3B、HDAC1等使miR-9-1发生表观遗传学沉默，解除了对AML1-ETO9a翻译水平上的抑制；4)促使其结合到重要的下游靶基因c-KIT的启动子区，并促进其异常表达，激活PI3K等信号途径，导致白血病的发生。5) 证实了SIRT1在AE阳性的细胞系和临床样本中是高表达的，并且高表达SIRT 1基因的样本中，其预后明显较差。6) 我们构建了SIRT 1启动子区的荧光素酶报道载体及其突变体，转染细胞，发现融合蛋白AE可以结合在SIRT1的启动子区，并且上调SIRT1的表达。ChIP实验证实，AE与SIRT1启动子区的结合。7) 同时，我们还发现，在AE阳性的临床样本中，HIF1α的表达明显比AE阴性的临床样本的表达高，HIF1α高表达的样本中，其预后明显比HIF1α低表达的差。这为阐明t(8;21)AML的发生和复发奠定了基础，也为白血病的诊断、预后和治疗提供新的手段和策略。