PcG蛋白EZH2在MDS表观遗传学发病机制中的作用及靶向干预研究

DNA methylation plays an important role in epigenetic inactivation of tumor suppressor genes (TSGs), which has been confirmed to be involved in tumorigenesis of MDS. Recently, biochemical evidence showed methylated lysine 27 of histone H3 (H3K27) was found to induce epigenetic silencing of TSGs independently of DNA methylation. However, the role of H3K27 methylation in MDS is still unclear. Our previous work showed that histone methyltransferase EZH2 that belongs to PcG (Polycomb group) family is overexpressed in MDS and positive in correlation with poor prognosis. The overexpression of EZH2 is also associated with downregulation of several TSGs. Therefore, we speculated that EZH2 as an potential oncogene takes part in tumorigenesis of MDS by regulating H3K27 methylation and inducing epigenetic silencing of TSGs. Based on the result of the previous studies, we will confirm our hypothesis by performing a series of experiments such as RNAi, ectopic expression, WB, ChIP, Pyrosequencing, DNA sequencing, etc. The purpose of this research was to verify the feature of EZH2 as an oncogene, investigate if EZH2 promotes malignant biological behavior of MDS by H3K27 methylation-induced silencing of TSGs and discuss the possibility of targeted intervention including biology and pharmaceutical treatment. Finally, this project would provide scientific evidence for novel targeted therapy or combination therapy, and then improve the therapeutic effect of epigenetic drugs.

DNA甲基化是抑癌基因表观遗传失活的一个重要途径，且已被证实参与了MDS病理过程。而组蛋白H3K27甲基化也被证实能够诱导抑癌基因失活，但是其在MDS中的作用尚无研究。申请人前期工作显示PcG蛋白EZH2(组蛋白H3K27甲基化转移酶)在MDS患者中高表达且与患者预后不良相关，且也与多个抑癌基因表达下调存在关联。我们推测EZH2作为一个潜在的癌基因，通过调控组蛋白H3K27甲基化来诱导抑癌基因沉默。本研究在已有工作基础上，采用慢病毒载体介导的RNA干扰技术、过表达技术、免疫印迹、ChIP、Pyrosequencing 甲基化定量分析等技术，验证EZH2在MDS中的癌基因特性，探讨EZH2是否通过甲基化H3K27诱导的抑癌基因沉默来调控 MDS克隆细胞恶性生物学特征，探讨影响EZH2表达和功能的生物学和药物学干预的靶向价值。该项目将为拓展新的表观遗传靶向治疗或联合治疗提供科学依据。

PcG蛋白EZH2介导的H3K27甲基化是正常细胞发育分化所必需的的，其异常是血液肿瘤发生关键原因。然而，EZH2在骨髓增生异常综合征发病(MDS)机制中的作用尚未明确。在本项目中，我们研究了EZH2在MDS患者中的表达水平及其致病机制。结果显示MDS患者呈现差异化的EZH2表达模式，低危患者尤其是伴有trisomy8的低危患者呈现相对高的EZH2表达，高危患者尤其是伴有单体异常的患者呈现明显低的EZH2表达水平。EZH2低表达预示着较差的生存以及较高的白血病转化。MDS患者7q36位点(EZH2)基因组拷贝数缺失 是导致EZH2低表达的关键原因。在体外试验中，EZH2敲低抑制MDS来源细胞株SKM-1细胞凋亡，促进细胞增殖和集落形成，明显增加SKM-1细胞的体外成瘤能力，同时导致缩减的H3K27me3水平。进一步H3K27me3的ChIP-on-ChIP试验结合患者EZH2表达揭示EZH2低表达导致广泛降低的H3K27me3水平，EZH2低表达导致HOX家族基因启动子区域H3K27me3水平降低。基因表达谱结合定量PCR显示EZH2低表达导致患者呈现增高的HOX(HOXA、HOXB、HOXC和HOXD)家族基因表达增加。最后的药物干预试验提示H3K27组蛋白甲基化抑制剂诱导MDS相关细胞系凋亡，但能诱导不同程度的HOX家族基因表达激活，可能与表观遗传修饰药物治疗后的药物耐药有关。这些研究探讨了EZH2在MDS中的功能特性，探讨EZH2介导的H3K27me3在癌基因激活的中的关键作用，丰富了MDS表观遗传发病机制，最后也探讨影响EZH2表达和功能的干预靶向价值。