【作者】 沈松菲；

【导师】 沈建箴；

【作者基本信息】 福建医科大学 ， 内科学， 2011， 博士

【摘要】 急性白血病是一个多因素相关的疾病。研究发现,急性白血病的发生、发展、治疗疗效及预后与异常的表观遗传学有关。表观遗传学(epiginetics)是研究不涉及DNA序列变化的、可遗传的基因表达的改变。它主要通过DNA甲基化、组蛋白翻译后修饰、RNA相关沉寂和染色质重塑等多种修饰机制调控基因的表达,是肿瘤发病的重要机制之一。目前研究较多的是单一的抑癌基因DNA甲基化和组蛋白乙酰化、组蛋白甲基化等修饰形式,对于不同修饰形式之间的相互作用及对基因表达的调控机制等研究尚不明确,是近年来表观遗传领域的研究热点。Wnt信号转导通路(wingless pathway)是一条保守的信号通路,参与调节细胞的生长、迁移、分化及凋亡,是肿瘤发生过程中的关键信号通路之一。研究发现,Wnt通路的异常活化与肿瘤的形成密切相关。本课题以Wnt通路拮抗基因为靶基因,拟通过双向干预DNA甲基化和组蛋白甲基化模式的平衡,探讨Jurkat细胞中DNA甲基化和组蛋白H3K9甲基化的相互作用及对靶基因的表达调控机制,研究其对Jurkat细胞生物学活性和Wnt信号通路传导的影响,揭示急性白血病的表观遗传学发病机制,挖掘新的靶分子,为白血病早期诊断和基因靶向治疗提供新的思路。课题分为以下四部分:1、以Western Blot方法检测急性白血病患者及7种恶性血液病细胞系的组蛋白H3K9、H3K4总甲基化水平,分析其与急性白血病发生、发展、治疗效果及预后之间的关系。2、以H3K9me3组蛋白高甲基化的急性淋巴细胞白血病Jurkat细胞系为研究对象,通过染色质免疫分析联合芯片(CHIP on chip)技术筛选出与H3K9me3甲基化位点相关的Wnt信号通路拮抗基因WNT5A、DKK3、SFRP2作为靶基因,qPCR法检测靶基因表达情况;并以甲基化特异性PCR(MSP)方法检测靶基因启动子区DNA甲基化水平,分析组蛋白甲基化和DNA甲基化两种表观遗传修饰机制之间的相关性,及与Wnt信号传导通路的联系。3、构建针对组蛋白H3K9me3位点甲基转移酶SUV39h1基因的干扰质粒转染Jurkat细胞,沉默该基因以逆转H3K9组蛋白甲基化;同时以经典的DNA去甲基化药物5-Aza-CdR去除靶基因启动区DNA甲基化,从DNA和组蛋白甲基化双向调控机制入手,以Western Blot、MSP、ChIP-qPCR等方法研究各自甲基化平衡模式被打破后,另一方对应的DNA/组蛋白甲基转移酶、DNA/组蛋白甲基化水平和靶基因表达的改变,探讨DNA甲基化和组蛋白甲基化之间的相互作用和对靶基因的表达调控机制。4、以未处理组为正常对照,研究5-Aza-CdR处理组和SUV39h1基因敲除组对Jurkat细胞的生长曲线、增殖活性、克隆形成能力、细胞周期以及凋亡水平等细胞生物学活性的影响,并以Western Blot法检测Wnt通路关键效应蛋白β-catenin的表达改变,探讨DNA/组蛋白甲基化修饰对Jurkat细胞生物学活性及Wnt信号通路的影响。结论如下:1.组蛋白H3K9me3高甲基化和组蛋白H3K4低甲基化在急性白血病中广泛存在,且与白血病的分型、治疗疗效和预后相关。2. Jurkat细胞组蛋白H3K9me3甲基化与Wnt信号通路拮抗基因(SFRP2、DKK3、WNT5A)启动区DNA的高甲基化状态有关。3. DNA去甲基化可引起组蛋白H3K9甲基化水平及组蛋白甲基转移酶SUV39h1和G9a的表达下调,激活靶基因表达;在体外能够抑制Jurkat细胞增殖,阻滞细胞周期进程,并诱导凋亡,其作用机制与通过抑制Wnt/β-catenin信号途径有关。4.组蛋白H3K9去甲基化,可引起DNA甲基转移酶DNMT1、DNMT3A和DNMT3B的表达下调,但对DNA甲基化无明显改变,靶基因表达增加;在体外能够抑制Jurkat细胞增殖,阻滞细胞周期进程,并诱导凋亡,其作用机制与通过抑制Wnt/β-catenin信号途径有关。5. DNA甲基化和组蛋白H3K9甲基化的失衡,可独立或协同激活Wnt通路拮抗基因的表达,其中H3K9组蛋白甲基化是优先于DNA甲基化的第一始动因素。更多还原

【Abstract】 Acute leukemia is a multiple factor relative disease. Some researches found that the acute leukemia occurrence, development, treatment effect and prognosis are relevant with epigenetics . Epigenetics is refered to the heritable gene expression variations without the DNA sequence changes. It is mainly through several mechanisms such as DNA methylation, histone posttranslational modifications, RNA silence and chromatin remodeling to moderate gene expression, which is also one of the important mechanism of tumour exist. Current researches mainly point on anti-oncogene DNA methylation, histone acetylation and histone methylation separately. It’s not clear to the interactions between these different modifications and the mechanisms of gene expression regulating, which in same time, are the recent focus of epigenetic research. Wnt signal pathway (wingless pathway) is a conservative signal pathway, involved in regulating cell growth, migration, differentiation and apoptosis. It is one of the most important signal pathway in tumor occurrence. The abnormal activation of Wnt pathway is closely related to the tumor formation.This project is planned to target the antagonist genes in Wnt pathway. By interfering up and down the balance between DNA methylation and histone methylation patterns, the results would be discussed on the interaction of Jurkat cell DNA and histone H3K9 methylation and on the regulation mechanism of target gene expression, including the research on the Jurkat cell lines biologic activities and its influence to Wnt signal pathway’s conduction, revealing the acute leukemia epigenetical pathogenesis, finding some new clues for the early leukemia diagnosis and gene targeting therapy.The project is combined of four parts:1. Adopting the Western Blot method, the total methylation levels of histone H3K9 and H3K4 were detected on acute leukemia patients and seven series of malignant leukemia cell lines, analyzing their relationships among the occurrence, development, treatment effect and the prognosis of acute leukemia.2. The acute lymphocytic leukemia Jurkat lines was set as the research object in which the histone H3K9me3 methylation level was high. ChIP-on-chip(chromatin immunopricipitation on chip) technic was used to screen the Wnt antagonism genes like WNT5A, DKK3 and SFRP2 as target genes. Then these target gene expressions were analyzed by qPCR method and their promoter DNA methylation levels by MSP method. The correlation between histone and DNA methylation and their relationships with Wnt were compared.3.Constructing interference plasmid for histone methyl transferase SUV39h1 on H3K9me3 site, silencing the SUV39h1 gene to reverse H3K9 histone methylation, at the same time, 5 - Aza– CdR as the classic DNA methylation drugs was used to wipe off the target gene promoter DNA methylation. Under the bi-regulating of DNA and histone methylation, methods including Western Blot, MSP and ChIP-qPCR were employed to detect the level changes of corresponding DNA/histone methyl transferase, DNA/histone methylation and target gene expression. The interaction between DNA and histone methylation and their regulation mechanism on target gene expression were then explored.4. As the non-drug group be normal control, the 5-Aza-CdR treatment group and SUV39h1 gene knockout group were cross compared in levels as its growth curves, proliferation activities, clone formation abilities, cell cycles and apoptosis. The express alteration of key effective proteinβ-catenin was detected by Western Blot. The influences of DNA/histone methylation modification on Jurkat cell’s biologic activity and Wnt signal pathway were discussed.Conclusions :1. The histone H3K9me3 high methylation and H3K4 low methylation are widely exist in acute leukemia and correlated to leukemia’s type, treatment effect and prognosis.2. Jurkat cell’s histone H3K9me3 methylation is correlated to promoter DNA hyper-methylation levels of Wnt signal pathway’s antagonism genes (WNT5A, DKK3 and SFRP2).3. The drug 5-Aza-CdR can reverse DNA methylation, down-regulate histone H3K9 methylation level, decrease histone methyl transferase SUV39h1 and G9a expression and activate the target gene expression.It also can inhibit Jurkat cell proliferation, block the cell cycle process and induce apoptosis in vitro. These effects may be due to inhibiting the Wnt/β-catenin signal pathway.4. SUV39h1 gene silence can reverse histone H3K9 methylation state, decrease the expression of DNA methyltransferase as DNMT1, DNMT3A and DNMT3B,increase the target gene expression, but there is no significant change of DNA methylation. It can inhibit Jurkat cell proliferation, block the cell cycle process and induce apoptosis in vitro. These effects may be due to inhibiting the Wnt/β-catenin signal pathway.5.The imbalance between DNA and histone H3K9 methylation can independently or collaboratively activate Wnt pathways antagonism genes expression, restrain Wnt signals. H3K9 histone methylation is a more earlier actived event than DNA methylation.更多还原