骨髓增生异常综合征中DNA去甲基化酶Tet基因调控Wnt/β-catenin信号通路的机制

Abnormal epigenetics is one of important pathogenesis of tumour. To investigate the pathogenesis of myelodysplastic syndrome (MDS), we focus on the relatonship of Tet gene family (Tet1,2,3) and Wnt signal pathway.We found some antagonism of Wnt pathway such as sFRP, DKK and SOX genes were aberrant hypermethylation in MDS and Wnt pathway was activated, which led to MDS progression. In recent years, Tet genes (tet1,2,3) have been found to have a new function that can regulate DNA methylated status by converting 5mC to 5hmC through hydroxylase activity. Our pilot study indicated lower expression of Tet gene was associated with the hyper-methylation of sFRP5, one of antagonism of Wnt pathway. This result suggested Tet gene maybe regulate the methylation of antagonism in Wnt pathway and activated the pathway. In this study, we will set up over expression and silence of Tet genes by transfection in MDS cell line, also generate Tet2 knockout mouse model. Dual lusiferase reporter system, RT-PCR and Western blot will be used to study whether Tet genes regulate the methylation of antagonism and activity of Wnt pathway. High performance liquid chromatography and mass spectrum and Chromatin Immunoprecipitation and DNA sequencing (ChIP-seq) are used to learn how Tet genes regulate Wnt pathway and the gene regulatory network.

为了探索骨髓增生异常综合征（MDS）的表观遗传学发病机制，对DNA去甲基化酶Tet1,2,3基因调控Wnt通路的机制进行研究。前期研究证实Wnt通路拮抗基因sFRP,DKK,SOX启动子甲基化在MDS患者中较常见，并可以导致Wnt通路激活，与疾病进展相关；Tet基因在正常人、MDS和急性髓系白血病(AML)患者中表达存在差异；Tet基因表达水平与sFRP5甲基化水平和Wnt通路下游基因表达水平相关，提示Tet基因可能通过改变Wnt通路拮抗基因的甲基化状态而调控Wnt通路。本课题将构建Tet基因过表达和沉默表达的MDS细胞株，建立Tet2基因敲除小鼠模型，采用荧光素酶基因报告、定量甲基化、Western blot等研究Tet基因如何通过调控Wnt通路拮抗基因甲基化状态而影响Wnt通路活性，采用高效液相色谱/质谱和染色质免疫沉淀测序 (ChIP-seq)研究Tet基因调控Wnt通路的机制。

DNA羟甲基化酶Tet在维持基因组甲基化状态的平衡、调控基因表达水平方面发挥重要作用，TET2基因突变与血液系统肿瘤发生关系密切；而临床标本的前期实验结果提示TET2基因表达与Wnt/β-catenin信号通路活性相关。为探索TET2基因在骨髓增生异常综合征（MDS）发病及进展中的生物学作用及其对Wnt/β-catenin信号通路活性的调控，本课题通过构建TET2基因沉默表达的MDS及AML细胞株模型以及Tet2基因敲除小鼠模型，进行细胞增殖、凋亡、周期等功能学检测及小鼠表型鉴定，并采用RT-PCR、Western blot和免疫荧光等技术检测Wnt/β-catenin信号通路活性的变化，从以下方面提供证据：（1）TET2基因沉默可显著增强细胞株SKM-1和THP-1的增殖能力，并可使细胞凋亡减少；（2）TET2基因沉默能够进一步激活Wnt/β-catenin信号通路，增强通路下游基因的转录表达；（3）Tet2基因敲除小鼠表现出CMML样和MDS样的病理特征，出现不同程度的骨髓造血抑制和衰竭；（4）Tet2基因敲除早期可导致Wnt/β-catenin信号通路的异常激活，而晚期通路则被抑制。综上，本研究从临床标本出发，通过细胞模型和动物模型进一步阐明TET2基因的生物学功能及其对MDS发病和进展的影响，深入探索TET2基因对Wnt/β-catenin信号通路的调控作用，为阐明MDS的部分表观遗传学发病机制，寻找新的临床药物治疗靶点提供线索。