【作者】 马爱妞；

【导师】 周向军； 王永祥；

【作者基本信息】 上海交通大学 ， 药理学， 2010， 博士

【摘要】 Survivin是凋亡抑制蛋白家族成员,具有调控有丝分裂和抑制细胞凋亡的作用。Survivin在大多数结束分化的正常组织中均未被检测到,但在几乎所有的肿瘤组织中均过量表达,并为肿瘤细胞生存所必须。因此,survivin被公认为是癌症治疗的一个重要靶点。研究发现,用一正义的短链甲基化寡核苷酸与某一基因启动子区序列互补,可以诱导该基因启动子区甲基化而抑制基因转录。利用这一机制来抑制致病基因的表达,应能起到治疗疾病的作用。据此,我们设计了一个与survivin基因启动子区域互补的22个碱基的甲基化脱氧寡核苷酸SurKex,在早期的工作中,我们通过体内外的药效学实验已经证明,SurKex能抑制survivin mRNA和蛋白的表达,并对荷瘤鼠肿瘤生长有抑制作用,且能延长荷瘤鼠的存活时间。同时,对SurKex作用机理也进行了初步研究,结果表明,它是通过诱导survivin启动子区域发生甲基化而导致基因沉默的。在前期工作的基础上,本文首先对SurKex诱导survivin启动子部位甲基化的作用方式进行了更深入的研究,通过亚硫酸氢钠测序法,确证了SurKex诱导survivin启动子部位发生甲基化的方式是靶向、定点诱导。i基因沉默时,与之结合的组蛋白往往会发生一些共价修饰的改变,所以接下来的研究,我们主要考察了SurKex诱导survivin沉默后,与survivin启动子区结合的组蛋白H3、H4上特定的赖氨酸残基的甲基化水平及H3、H4的乙酰化/去乙酰化水平是否发生相应的改变。我们采用染色质免疫沉淀技术,通过特异性抗体进行分析,结果表明,SurKex作用后,与survivin启动子区结合的组蛋白H3-K9二甲基化、H3-K27三甲基化水平升高;H4乙酰化、H4-K16乙酰化水平降低,也即去乙酰化水平升高。组蛋白的这些改变,与基因沉默是呈正相关的。DNA甲基化和组蛋白的共价修饰,都是调控基因表达的重要表观遗传学生化事件。关于二者之间的因果关系,进行调控的先后顺序,一直是表观遗传学领域研究的热点。在我们的实验中,SurKex作用后,既引起DNA甲基化,又引起组蛋白尾部修饰的改变。它们之间是如何彼此协作、控制survivin的开或关,更确切地说,是DNA甲基化指导组蛋白共价修饰的改变,还是组蛋白共价修饰的改变指导DNA甲基化,是我们接下来的研究要解决的关键问题。我们选用DNA甲基化酶抑制剂5-氮脱氧胞嘧啶核苷(5-Aza-CdR)、组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)和组蛋白甲基化酶抑制剂BIX-01294,分别考察DNA甲基化或组蛋白去乙酰化或甲基化作用被相应抑制剂阻断后,对SurKex效应的影响,以此阐明DNA甲基化和组蛋白共价修饰(去乙酰化和甲基化)在survivin沉默中的因果关系和调控顺序。具体方法是通过RT-PCR考察survivin mRNA表达情况、Bisulfite sequencing分析survivin启动子区特定CpG甲基化情况及染色质免疫沉淀(ChIP)实验考察与survivin结合的组蛋白共价修饰情况。实验结果表明,DNA甲基化是survivin沉默中最关键的一步,但组蛋白去乙酰化和甲基化指导DNA甲基化,它们的作用途径可能与DNA甲基化转移酶1(DNMT1)有关。最后,我们想初步验证对上述实验结果的推论是否正确。我们用TSA和BIX-01294分别阻断组蛋白去乙酰化和甲基化后,用RT-PCR考察对DNMT1 mRNA表达的影响。结果表明,组蛋白去乙酰化和组蛋白甲基化被相应的抑制剂阻断后,都出现了DNMT1 mRNA表达的降低。由此可见,组蛋白去乙酰化和甲基化都可能通过上调DNMT1 mRNA表达而影响DNA甲基化。当然,这可能只是它们作用途径中的一个环节。本课题的研究首先确证了SurKex为一种新型的靶向、定点甲基化诱导剂,它通过诱导survivin基因启动子区特定CpG位点甲基化而引起survivin沉默;其次阐明了SurKex在诱导survivin沉默的过程中,除了DNA甲基化之外,组蛋白H3-K9二甲基化、H3-K27三甲基化水平升高,组蛋白H4、H4-K16乙酰化水平降低;接着,以SurKex为工具药,初步阐明了在survivin沉默过程中,DNA甲基化是最关键的一步,但组蛋白共价修饰的改变(组蛋白去乙酰化和甲基化)是更优势的事件,它指导DNA的甲基化;最后,初步证明组蛋白共价修饰的改变(组蛋白去乙酰化和甲基化)是通过影响DNMT1 mRNA的表达,来指导DNA甲基化。本课题的研究不但为抗肿瘤药物的设计提供了新的思路,提供了从表观遗传学角度靶向治疗癌症的一种新方法,而且为解决表观遗传学领域的重大理论问题提供了实验证据。更多还原

【Abstract】 Survivin is an inhibitor of apoptosis protein (IAP) family member, it is the apoptosis inhibitor as well as the mitotic regulator. Survivin is absolutely undetectable in normal tissues, but over-expressed in all the most common human cancers. Also, it is required for maintaining cancer-cell viability. Thus, Survivin is considered as a therapeutic target in cancer.According to previous research, in mammalian cells, a short methylated sense oligonucleotide that is complementary to the sequence of the promoter region of a given gene can induce hypermethylation of the promoter region of that gene and thus inhibit that gene transcription. This mechanism might be used to cure diseases by inhibiting the expression of a certain gene harmful to health. By virtue of this, we designed a methylated oligodeoxynucleotide (SurKex, 22 bases) complementary to a region of part promoter of survivin. Our early experiments showed that SurKex can inhibit not only survivin mRNA and protein expression, but also tumor growth of tumor-bearing mice, and prolong survival of animal model of nude mice by pharmacodynamics in vivo and in vitro. At the same time, we had a preliminary study on the mechanism of SurKex. The results showed that SurKex could lead survivin gene silencing by inducing DNA methylation of survivin promoter region.In this article, at the basis of our previous research, we first had a further study on methylation mode of survivin promoter inducing by SurKex through bisulfite-sequencing and confirmed that SurKex can lead to methylate survivin promoter in CpG-site specific way, that is targeting induction.When gene silencing, some changes of covalent modifications of histone combined with a certain gene will accordingly happen. Next, we used chromatin immunoprecipitation (ChIP) to investigate the modifications of acetylation/deacetylation or methylation on the specific lysine residue of histone H3, H4 combined with survivin promoter sequence after survivin promoter site-specific methylation was induced by SurKex. The results showed that SurKex increased histone H3-K9 dimethylation、H3-K27 trimethylation and decreased H4、H4-K16 acetylation levels. These changes of histone modifications positively related with gene silencing. Although DNA methylation and histone covalent modifications are both important biochemical events of epigenetics in regulating gene expression, there is a hot debate in epigenetics about the order and the causal relationship between them when they regulate gene expression. In our experiment, SurKex not only induced methylation of survivin promoter but also changes of histone tail modifications. Now that how did they collaborate with each other on survivin expression and control survivin“on or off”precisely? Whether DNA methylation guides histone covalent modifications or histone covalent modifications guide DNA methylation would be a key issue to be solved in our next study.Then we studied the effects on the efficacy of SurKex in blocking DNA methylation, histone deacetylation or histone methylation by DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-Aza-CdR), histone deacetylase inhibitor trichostatin A (TSA) and histone methyltransferase inhibibor BIX-01294 respectively. We determined the expression of survivin mRNA by RT-PCR, analyzed the site-specific CpG methylation of survivin promoter region by bisulfite sequencing and detected the covalent modifications of histone tails combined with survivin promoter by chromatin immunoprecipitation (ChIP). These results showed that DNA methylation was the crucial step in survivin silencing, but histone deacetylation and methylation guided DNA methylation, which could play a role through DNMT1.Finally, we would be in an attempt to verify whether our inference was correct or not. We investigated the expression of DNMT1 mRNA by RT-PCR in blocking histone deacetylation and methylation by TSA and BIX-01294 respectively. The results showed that the expression of DNMT1 mRNA was decreased greatly after histone deacetylation or histone methylation being blocked, which revealed that both of them could affect DNA methylation by upregulating the expression of DNMT1 mRNA. Of course, this is only a possible means of their roles.In summary, we first confirmed that SurKex is a new type of targeted, site-directed CpG DNA methylation drug candidate, and it could result in survivin silencing by inducing site-specific CpG methylation of survivin promoter region; Next we revealed that SurKex could increase histone H3-K9 dimethylation and H3-K27 trimethylation levels and decrease histone H4 and H4-K16 acetylation levels besides DNA methylation; Then we verified that DNA methylation is a vital step in survivn silencing, but the change of histone covalent modifications (including histone deacetylation and methylation) is more advantage event, and it guide DNA methylation; Finally, we initially proved that histone deacetylation and methylation guide DNA methylation by affecting the expression of DNMT1 mRNA.This research provides not only new ideas for the design of antineoplastic drugs and a new method for cancer targeted therapy from cancer epigenetics, but also the experimental evidence for solving the major theoretical problem in the field of Epigenetics.更多还原