【作者】 张广华；

【导师】 李强； 江世文；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2010， 博士

【摘要】 研究背景：子宫内膜癌在我国是最常见的妇科肿瘤之一,威胁到很多女性的健康与生命。值得注意的是,一些研究表明虽然子宫内膜癌的发病率基本稳定,但死亡率似有上升趋势。我们面临的最大挑战是如何提高病人的生存率。孕激素治疗子宫内膜癌的作用已经得到公认,但是晚期肿瘤中孕激素受体表达经常缺失,导致其对孕激素的敏感性降低。因此孕激素受体阴性肿瘤对孕激素治疗的低敏感性是目前临床激素治疗的重要课题。虽然已经知道孕激素受体表达缺失可能与孕激素受体基因DNA的异常甲基化有关,但其中具体的分子机制并不清楚。不知道异常甲基化是否起决定性的作用,也不清楚究竟在孕激素受体基因沉默的过程中其染色质上发生了什么变化。搞清楚这些途径不但有助于对于增进子宫内膜癌的治疗,而且可以帮助我们更好地理解表观遗传学调控的一般机制。目的：本课题将对子宫内膜癌细胞系中染色质成分改变与孕激素受体基因转录沉默之间的关系进行研究。具体来说进行以下的四大部分实验：1)确定PR-B在几种细胞系中的表达与甲基化状态；观察DNA甲基转移酶(DNMT)及组蛋白乙酰化酶(HDAC)抑制剂对孕激素受体基因重新激活作用,找出最佳的时间与浓度。2)确定孕激素受体染色体成份的改变与孕激素受体表达的关系,逐个检测MBD蛋白及组蛋白修饰在孕激素受体基因表达中的作用。3)观察MBD及组蛋白表达水平与孕激素受体表达之间的关系。4)进一步研究MeCP2在孕激素受体表达控制中的作用。方法：首先测定了十八种子宫内膜癌细胞系中孕激素受体的表达水平,最终选定了孕激素受体表达阳性和阴性的细胞系作进一步的研究。采用甲基化特异性PCR的方法确定了孕激素受体的甲基化状态,并使用染色体沉淀(Chromatin Immunoprecipitation, ChIP)的方法鉴定孕激素受体基因染色体上甲基化DNA结合蛋白和组蛋白翻译后修饰在不同细胞、不同基因表达状态下的状况,以及在药物处理前后的改变。结果：发现多种DNA结合蛋白,包括MeCP2, MBD1, MBD2, MBD3, MBD4在甲基化／表达沉默和非甲基化／表达活跃的基因中呈现完全不同的结合状态,用DNA甲基化转移酶和组蛋白乙酰化酶抑制剂处理后的细胞可引起MBD结合和组蛋白修饰的明显改变。具体来说：1)在KLE和HEC1B细胞甲基化的PR-B启动子区检测到MeCP2的结合。相反,在Ishikawa细胞非甲基化的启动子区未发现MeCP2结合。2) MBD1和MBD2能结合甲基化和非甲基化的PR-B启动子区。用ADC处理可以诱导MBD1与甲基化的(而非未甲基化)PR-B启动子区的结合。同样的处理可以在KLE细胞中诱导MBD2的结合,而在HEC-1B或者Ishikawa细胞中没有观察到此作用。3)在任何细胞系都没有发现有MBD3和MBD4的结合。4)TSA处理在任何一个细胞系都未能改变MBD的结合状态。因此,不同的MBD家族成员表现出差异的和动态的与PR-B启动子相互作用的状态。接下来用ChIP方法检测PR-B启动子区H3 H4乙酰化和H3赖氨酸9位甲基化和赖氨酸4位甲基化。我们观察到：1)在三个细胞系中比较总的H3 H4结合水平,甲基化的PR-B启动子区比非甲基化的PR-B启动子区H3和H4的乙酰化水平明显低。2)使用ADC或者TSA处理会明显的增加甲基化的PR-B启动子区组蛋白乙酰化水平。3)H3-K9甲基化水平在甲基化的PR-B启动子区比非甲基化的PR-B启动子区升高,而H3-K4甲基化显示与其相反。4)用ADC和TSA处理KLE细胞,结果H3-K9明显去甲基化,但却能诱导H3-K4甲基化。5)与以上在甲基化的PR-B启动子区的发现相反,ADC和TSA处理没有明显的影响非甲基化的PR-B启动子区组蛋白乙酰化和甲基化水平。结论：通过这一研究,认识到H34位赖氨酸上的低甲基化和H39位赖氨酸上的高甲基化是表达沉默的孕激素受体基因启动子的特点。经DNA甲基化转移酶和组蛋白乙酰化酶抑制剂处理后MeCP2的结合及组蛋白修饰可以发生逆转,导致孕激素受体基因的重新激活。免疫印迹检测表明细胞内整体水平上的蛋白浓度保持不变,说明这些改变特异性发生于孕激素受体基因的局部,而与药物在细胞整体的作用无关。为了进一步证实MeCP2在孕激素受体基因表观遗传学中的重要作用,我们进行了siRNA基因表达敲除实验。结果表明MeCP2对甲基化启动子的结合对孕激素受体基因表达沉默至关重要。在MeCP2表达敲除的细胞中,MeCP2对孕激素受体基因的结合降低,于此相关联,孕激素受体基因表达沉默发生部分逆转,引起孕激素受体mRNA的上升。这些发现阐明了孕激素受体基因表达调控的一些重要特点与机制,为近年来这一领域的首创,对于明确孕激素受体基因的表观遗传学提供了重要的信息。这些发现将有助于设计新的表观遗传干预方法以重新激活孕激素受体的表达,从而增进孕激素受体阴性的子宫内膜癌对激素治疗的敏感性。比如说,可以使用甲基化转移酶抑制剂(如ADC)和组蛋白去乙酰化酶抑制剂(如TSA)对孕激素受体肿瘤阴性病人进行预处理,等孕激素受体表达后再给予大剂量孕激素杀死肿瘤细胞。因为多数死于晚期或手术后复发的子宫内膜癌病例的孕激素受体为阴性,所以这种治疗方法有巨大的发展前景。根据此次研究的成果我们可以设计动物实验对这一治疗模型进行进一步验证。更多还原

【Abstract】 Background:Endometrial carcinoma is the most common gynecologic malignancy threatening many women’s health and lives. It is noteworthy that results from some studies indicated while the incidence rate appears to be stable, the death rate is rising. The challenge we are facing is how to increase the survival rate and extend the life of patients. The therapeutic value of progestational treatment for endometrial hyperplasia and early stage cancers has been well recognized. However, in advanced and recurrent cancers the progesterone receptor is often lost, leading to a decreased sensitivity to progestational therapy. Therefore how to improve the efficacy of progestins for treating advanced and recurrent cancers has become an important topic. While aberrant DNA methylation of a CpG island in PR-B promoter is considered the major cause for PR negativity, the epigenetic mechanism has not been not well understood. It is not clear if DNA methylation play a decisive role, and what occurs in the chromatin during the epigenetic silencing of progesterone receptor gene. Better understanding of the pathway will not only help us to improve the treatment of endometrial cancer, but also help us to understand the epigenetic mechanism controlling gene expression.Objective:In this study, we determine the specific changes in chromatin composition that are associated with the PR-B transcriptional silencing in endometrial cancer cells. We performed for parts of studies:1) to confirm the PR-B expression status in several endometrial cancer cell lines and observe the reactivation of PR-B transcription by DAN methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors.2) To determine the relationship between PR-B expression and epigenetic silencing. Using chromatin immunoprecipitation assay, we compared the methyl CpG-binding proteins (MBD) binding and histone modification alterations among cells with different PR-B methylation and expression status, as well as cells before and after treatment with DNA methyltransferase or histone deacetylase inhibitors.3) To observe the relationship between MBD binding/histone modification PR-B expression.4) To further investigate the role of MeCP2 in the expression control of PR-B. Methods:We first determined the PR expression status in 18 different endometrial cancer cell lines, and chose PR-B negative and positive cell lines for further analysis. We applied methylation specific PCR to determine the methylation status of PR-B in these cells. Chromatin Immumoprecipitation (ChIP) assay was used to examine the MBD binding and histone posttranslational modification in the PR-B chromatin from different cells with different PR-B expression patterns, or cells before and after drug treatment.Results:We have found methyl CpG-binding proteins, including MeCP2, MBD1, MBD2, MBD3, and MBD4 exhibit diverged binding profiles for methylated/silenced and unmethylated/active progesterone receptor gene. In addition, treatment with DNA methyltransferase and histone deacetylase inhibitors causes dynamic changes in both MBD binding and the histone modifications within chromatins associated with progesterone receptor gene.1) In KLE and HEC-1B cells with methylated PR-B gene, MeCP2 was found to bind to PR-B promoter. In contrast, no MeCP2 binding was observed in the Ishikawa cells in which PR-B gene is unmethylated.2) MBD1 and MBD2 bind to both methylted and unmethylated PR-B gene. ADC treatment induced the MBD1 binding of methylated, but not unmethylated PR-B promoters. The same treatment induced MBD2 binding in KLE cells.3) No change was found in the binding of MBD3 and MBD4.4) TSA treatment did not lead to alteration in MBD binding. Therefore, different MBD family members display varied binding pattern and dynamics upon their binding to PR-B gene.We also used the ChIP assay to examine the H3 and H4 acetylation and methylation at K4 and K9A positions. We found that 1) H3 and H4 acetylation is at their low levels in methylated PR-B promoters when compared to unmethylated promoter.2) ADC and TSA significantly affect the H3 and H4 acetylation levels.3) H3-K9 methylation level in the methylated PR-B is higher than in unmethylated PR-B. The H3 K4 methylation showed an opposite pattern.4) ADC and TSA treatment of cells led to decreased methylation of K9 but increased methylation in K4 position.5) In contrast to the findings in the methylated PR-B gene, ADC and TSA treatment had no significant effect on the histone acetylation and methylation in the unmethylated PR-B gene. Conclusions:We found that the low levels of histone H3 K4 methylation and high levels of K9 methylation is the characteristics of the methylated and silenced PR-B promoter. Treatment of PR-B negative cells with DNA methyltransferase and histone deacetylase inhibitors resulted in reversal of the MeCP2 occupancy and histone covalent modifications, and reactivation of the silenced PR-B gene. Importantly, Western blot analysis showed that MBD expression levels remain unchanged following treatment with DNA methyltransferase and histone deacetylase inhibitors. Thus, the observed changes in the chromatin composition are not caused by global drug effects, rather, they are most likely mediated by specific action associated with local changes on PR-B gene. To further confirm the important role of MeCP2 in epigenetic regulation of PR-B, we performed siRNA-mediated MeCP2 knockdown and measured the MeCP2 binding to PR-B promoter and PR-B mRNA levels. These experiments demonstrated that MeCP2 binding to methylated PR-B gene is critical for PR-B silencing.These findings have for the first time, provided new insights into the epigenetic mechanism leading to PR-B silencing in endometrial cancer. The information will be useful for the design epigenetic interference for reaction of PR-B, and enhancement of the sensitivity of endometrial cancer cells to hormonal therapy. For example, we may be able to more efficiently kill the cancer cells by pretreatment of cancer patients with DNMT and HDAC inhibitors. Since the advanced and recurrent cancer cause most of deaths of patient and many of these cancers are R-B negative, this strategy appears to be highly attractive. Using the current data, we will be able to design animal model to test the efficacy of this new treatment modality.更多还原