缺氧对急性髓系白血病DNA甲基羟化酶TET2的影响及机制研究

Hypoxia is involved in regulation of epigenetic modification, and even epigenetic modification is thought as a crucial player in the hypoxia reaction. However, its role in acute myeloid leukemia(AML) is not completely understood. The AML CD34+ primary cells and KG1a cell lines will be used for study the global genome DNA methylation patterns and specific genes (ID4 and P15) methylation status. Then, the recombinant DNA technique is used to observe the regulation of HIF-1α to the expression and function of TET2 in AML. Furthermore, luciferase reporter gene, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay are used to explore whether TET2 is a novel target gene that is transcriptionally regulated by HIF-1α. In this study we investigated the influence of the hypoxic on DNA methylation patterns in AML and the potential interaction between HIF-1α and TET2 as a potential mechanism,which will establish a molecular link between hypoxia and genomic DNA methylation in AML.

缺氧参与表观修饰调节，甚至有学者认为表观修饰调节在低氧反应中发挥关键作用，但在急性髓系白血病中相关机制并不清楚。本课题以人急性髓系白血病AML CD34+原代细胞及细胞系KG1a为研究对象，从整体基因组甲基化水平及具体基因甲基化态势两方面观察缺氧对白血病细胞甲基化表观修饰的影响；并采用基因重组技术，从缺氧诱导因子HIF-1α过表达及表达抑制两方面研究HIF-1α对白血病细胞内DNA羟甲基化酶TET2 表达及功能的调节，进一步采用蛋白与DNA结合试验及转录活性检测对相关调节机制进行探讨，以明确缺氧对白血病细胞基因甲基化的作用及潜在机制，为临床白血病诊断及治疗领域相关靶向分子的寻找提供实验依据。

缺氧参与白血病的表观修饰调节。本课题以人急性髓系白血病细胞系KG-1及AML 患者骨髓单个核细胞为研究对象，首先通过CCK-8实验、LDH测定及Annexin V-FITC/PI双标流式法观察不同氧浓度及缺氧时间对AML细胞增殖及凋亡的影响，结果发现：缺氧促进AML细胞增殖、抑制凋亡。随后通过构建HIF-1α过表达质粒及采用HIF-1α抑制剂YC-1，从HIF-1α过表达及表达抑制两方面研究其对白血病细胞内DNA羟甲基化酶TET2 表达的影响及功能的调节，结果发现：缺氧通过上调转录因子 HIF-1α的表达进而促进TET2表达、基因组去甲基化（5-hmC水平）和癌基因P15去甲基化。进一步采用染色质免疫共沉淀及荧光素酶报告基因实验发现HIF-1α通过与TET2基因启动子区缺氧反应原件结合而增强其转录活性。本研究发现TET2为HIF-1α调节的潜在靶基因，并且缺氧可以通过HIF-1α对TET2的表达进行调控，进而影响下游靶基因的甲基化及表达，在白血病发生及进展过程中发挥分子生物学作用。本研究对AML中缺氧代谢与基因表观调节的关系进行了初步探讨，为急性白血病等血液系统恶性肿瘤的诊治研究提供了新的思路与实验依据。项目资助发表SCI论文1篇，待发表SCI论文1篇。培养硕士生2名。