【作者】 翟荣林；

【导师】 王国斌；

【作者基本信息】 华中科技大学 ， 外科学， 2008， 博士

【摘要】 研究目的:了解结肠癌细胞中雌激素信号传导通路(Estrogen Signaling)的异常甲基化模式以及其与转录失活的因果关系,探讨雌激素受体β亚型基因(ERβ)对雌激素信号传导通路的表观遗传学调控方式,从不同层次上深入研究ERβ基因衰老性甲基化的表观遗传学机制及其病理意义,了解ERβ基因甲基化在结肠癌早期诊断、预防、预后评估以及治疗中的应用价值,尤其是雌激素抑制结肠肿瘤发生的作用机制,探索将此抑制作用用于已发生的肿瘤的治疗过程的应用价值和方法。研究方法:在整个一系列实验过程中,作为预实验部分,我们首先采用甲基化特异性PCR(MSP)方法了解了结肠癌细胞株(HT-29,Caco-2和Lovo)中ERβ及其靶基因PR(含经典的ERE元件)和c-fos原癌基因(含非经典的AP-1结合位点)启动子CpG岛的甲基化模式,采用常规PCR方法检测了结肠癌细胞株中ERβ基因、hMLH1基因、DNA甲基化转移酶1(Dnmt1)、DNA甲基化转移酶3a(Dnmt3a)、DNA甲基化转移酶3b(Dnmt3b)以及组蛋白甲基化转移酶(HMT)等表观遗传学酶谱的表达水平,为后续的研究打下基础。在此基础上,我们采用针对ERβ基因的RNAi技术体外抑制ERβ基因的表达,观察了ERβ基因转录失活对PR和c-fos基因启动子甲基化模式的调控以及表观遗传学酶谱表达的影响;同时,采用雌激素诱导或外源性ERβ基因导入等不同手段,正向调节ERβ基因表达,观察ERβ基因表达恢复或激活对雌激素信号传导通路靶基因启动子甲基化模式的调控以及表观遗传学酶谱表达的影响。通过对ERβ基因正反两个方向的表达调节,观察除了经典的受体-配体结合方式外,ERβ基因是否还存在表观遗传学调控方式或机制。接下来的研究中,我们采用“甲基化特异性寡核苷酸技术”,设计并合成针对ERβ基因的特异性甲基化寡核苷酸片段,作用于原代培养的雌性乳鼠结肠上皮细胞中,以诱导ERβ基因启动子发生特异性甲基化而失活,从而体外构建ERβ基因衰老性甲基化的结肠上皮细胞模型,以期模拟或者还原ERβ基因在结肠癌细胞中的表型和基因型,并在此基础上观察ERβ基因衰老性甲基化对雌激素信号传导通路的表观遗传学调控效应,深入探讨ERβ基因衰老性甲基化的表观遗传学机制及其病理意义,尤其是在雌激素预防女性结肠癌发生中可能的作用机理,为研究结肠癌的发病机制、预防和早期诊治结肠癌提供相关理论依据和实验基础。研究结果:雌激素信号传导通路相关基因中,ERβ及其靶基因PR启动子CpG岛在结肠癌细胞株HT-29、Lovo和Caco-2中均呈现为典型的异常高甲基化模式;原癌基因c-fos启动子CpG岛在三株结肠癌细胞中则表现为不完全性的部分甲基化。RT-PCR检测结果显示,与内参基因相比,ERβmRNA在三株结肠癌细胞中均呈现为弱表达状况。细胞爬片的免疫组织化学检测结果显示,ERβ蛋白主要定位在胞核,胞浆也有少量表达,其中在Caco-2中呈现中低度表达状况,在Lovo和HT-29中则表现为弱表达。总的来说,无论是ERβmRNA还是ERβ蛋白在三株癌细胞株中的表达都是被抑制的。hMLH1基因mRNA在三株结肠癌细胞中的表达无显著差异,均表现为中度表达水平。Dnmt1、Dnmt3a、Dnmt3b和HMT在结肠癌细胞株中呈现不等的表达水平,Dnmt3a、Dnmt3b在分化良好的癌细胞中呈现过度表达状况,而在分化不良或未分化的癌细胞中则被抑制表达。针对ERβ基因设计的特异性小干扰RNA片段(siRNA)作用于Caco-2后,ERβ基因mRNA表达显著下调;然而,Caco-2细胞中ERβ,PR和c-fos等基因启动子甲基化模式在RNAi前后却无任何改变;同时,对Dnmt1,Dnmt3a,Dnmt3b,HMT和hMLH1基因mRNA的检测也发现,RNAi作用前后这些基因的表达无显著改变。终浓度分别为10-6mol/L、10-8mol/L的雌二醇(E2)分别作用于Caco-2细胞两周后,ERβ基因表达无明显增高或其他改变。提示,单纯应用E2作用癌细胞可能并不能诱导ERβ的表达。接下来的研究采用常规基因重组技术构建ERβ真核表达载体并导入结肠癌细胞Caco-2。结果表明,外源性ERβ基因的导入可以显著的激活雌激素信号传导通路基因(PR、c-fos)的表达(P<0.01),与此同时,该通路基因总体甲基化密度显著降低。结合甲基化酶谱(Dnmt1,Dnmt3a,Dnmt3b和HMT)表达水平的改变,我们推测,这种调控作用很可能是通过去甲基化机制来实现的。不过,因为并没有完全逆转原有的高甲基化模式,因此,ERβ对PR、c-fos的调控作用很可能是但又不完全是通过去甲基化机制来实现的。换句话说,结肠癌细胞中ERβ基因激活可以部分地逆转雌激素信号传导通路基因的甲基化模式,使PR基因(含经典的ERE元件)和c-fos原癌基因(含非经典的AP-1结合位点)发生部分去甲基化并恢复近似正常的表达,在此过程中ERβ基因本身也发生了去甲基化改变。此外,ERβ基因导入Caco-2后,hMLH1表达也明显降低。ERβ与hMLH1之间是否存在以及存在何种相关性,尚有待进一步研究。我们成功的进行了SD新生大鼠结肠上皮细胞的体外原代培养,免疫组织化学检测表明,上皮细胞纯度达95%以上。我们针对ERβ基因设计合成的特异性甲基化寡核苷酸(MO)片段成功转染进入了结肠上皮细胞,并且在增殖比较旺盛的细胞团块中富集。MSP检测结果表明,转染进入细胞的MO片段成功诱导了ERβ基因启动子的特异性甲基化。结肠上皮细胞ERβ基因衰老性甲基化模型的建立,为从表观遗传学角度研究ERβ基因在结肠癌发生机制中的作用奠定了坚实的基础。深入研究发现,ERβ基因发生衰老性甲基化而灭活的同时,下游的靶基因PR和c-fos的表达也显著降低。对表观遗传学酶谱的检测则表明,MO诱导前后从头甲基化酶(Dnmt3a,Dnmt3b)和组蛋白甲基化酶(HMT)的表达也发生了显著的改变,维持甲基化酶(Dnmt1)的表达却无明显变化。这表明,整个过程中发生了显著的酶促条件下的从无到有的基因高甲基化和组蛋白高甲基化等表观遗传学基因型改变。结合正常结肠上皮细胞中该通路非甲基化或低甲基化这一事实,就更容易理解上述改变。研究结论:我们的研究结果提示,在结肠上皮细胞中,除了经典的受体-配体结合方式外,ERβ基因还存在着表观遗传学作用方式和机制,作为结肠上皮细胞中的优势受体,ERβ基因的衰老性甲基化,对雌激素信号传导通路中相关基因启动子甲基化模式及正常的表达水平起着决定性作用:ERβ基因启动子衰老性甲基化→ERβ基因表达灭活→表遗传学酶谱活性发生改变,下游的PR,c-fos等基因启动子发生异常高甲基化→PR,c-fos等下游靶基因表达失活→正常的雌激素信号传导通路发生异常→结肠上皮细胞增殖异常,MSI和CIMP表型改变,最终促进癌变?我们的研究有助于探讨ERβ基因的衰老性甲基化现象在结直肠肿瘤发生过程中的意义,特别是对结肠上皮的表观遗传学影响,了解ERβ基因衰老性甲基化在结肠癌早期诊断、预防、预后评估以及治疗中的应用价值,我们的研究还有助于明确雌激素抑制结肠的肿瘤发生的作用机制,探索将此抑制作用用于已发生的肿瘤的治疗过程的应用价值和方法。更多还原

【Abstract】 Objectives:It is indicated that estrogen receptor (ER) gene may play an important role in the process of modifications of histone tails and nucleosome remodeling by recruiting transcription factors such as Histones acetyltransferases (HAT) and Histone methyltransferase(HMT),which suggests that ERβgene can also mediate epigenetic regulation on its downstream targeted genes and consequently participates in carcinogenesis except for the classical signaling pathway via estrogen reaction element(ERE).Then how does it perform this process in colon cancer?Since promoter hypermethylation could result in transcription inactivation and transcription inactivation could mediate epigenetic regulation on its downstream targeted genes,then is there a cause and effect relationship between promoter hypermethylation of ERβgene and its epigenetic regulation?In other words, Does whether aging hypermethylation of ERβpromoter trigger the epigenetic regulation on its downstream targeted genes or not? This research is just executed to try to answer these questions and to know about pathologic mechanism of ERβgene aging hypermethylation and further the pathogenesis of the female ascending colon caner.Methods:Promoter CpG islands methylation modes of ERβ, progesterone receptor (PR) and c-fos genes were determined by methylated specific PCR(MSP) and the mRNA levels of ERβ,DNA Methyltransferase(Dnmt) 1, Dnmt3a,Dnmt3b, Histone Methyltransferase(HMT) and hMLH1 genes were detected by RT-PCR in HT-29、Lovo and Caco-2 colon cancer cells,separately. The expression level of ERβgene was artificially down-regulated using small interfering RNA(siRNA) and up-regulated by treatment with estradiol or ERβeukaryotic expression vector,separately, to determine the modulating action on estrogen signaling by ERβgene and the possible mechanism in Caco-2 cells.For the first time, we have established a new functional primary culture cell model by inducement of ERβgene promoter aging hypermethylation in Sprague Dawley rat normal colonic epithelial cells using a novel methylated oligonucleotides(MOs).Suckling rat colonic epithelial cells were primary cultured in DMEM media. Two MOs complementary to promoter regions of ERβwere synthesized and used on the cultured cells to induce promoter hypermethylation of ERβgene.MSP method was used to determine the methylation status of ERβpromoter and RT-PCR was used to detect the expression abundances of ERβmRNA in the cultured cells after MOs treatment.To study the epigenetic regulation on downstream target genes by aging hypermethylation of ERβgene,the expression levels of PR,c-fos,hMLH1,and epigenetic enzymatic spectrum genes,such as Dnmt1,Dnmt3a,Dnmt3b and HMT,were detected by RT-PCR in the cell model 48hours after MOs treatment.Results:1.The ERβand PR genes promoter were hypermethylated in HT-29、Lovo and Caco-2 colon cancer cells(P<0.01), whereas the c-fos gene promoter which contained the AP-1 site, partly showed a methylated CpG islands in all the three cancer cells(P<0.05).2.It was indicated that significant overexpression of Dnmt3b was seen in the human colon cancer cells while Dnmt1, HMT and hMLH1 genes were only modestly overexpressed.Dnmt3a and ERβgenes showed a very low expression level in all the three cancer cells. A significant discrimination of Dnmt3b mRNA expression levels was found between HT-29、Lovo and Caco-2 cells because Dnmt3b was present at high level in HT-29 and Lovo cell lines but declined drastically in Caco-2 cells.3.siRNA targeted ERβgene in Caco-2 cells could not change promoter hypermethylation modes of ERβ,PR and c-fos genes as well as the expression levels of Dnmt1,Dnmt3a,Dnmt3b and HMT genes.It seemed to have no effect on estrogen signaling pathway by down-regulating ERβgene expression in Caco-2 cells.4.Caco-2 cells were treated with 10-6mol/L or 10-8mol/L estradiol for 2 weeks to induce activation of ERβgene,but it failed.ERβeukaryotic expression vector was constructed successfully and then transfected into Caco-2 cells to reactivate ERβexpression. The mRNA levels of PR,c-fos,Dnmt3a and Dnmt3b gene were up-regulated in Caco-2 cells transfected with the recombinant compared with the control(P<0.01). The mRNA levels of Dnmt1,HMT and hMLH1 gene were down-regulated in Caco-2 cells transfected with the recombinant(P<0.01). At the same time,ERβ,PR and c-fos genes displayed an incomplete demethylating modifications in Caco-2 cells transfected with the recombinant.5.Suckling rat colonic epithelium cells was successfully cultured in vitro.Identification by immunohistochemical staining showed that more than 95% of the cultured cells were epithelial. The methylated and unmethylated oligonucleotides(UMOs) we designed and synthesized were transfected into the colonic epithelium cells successfully and assembled in the nucleolus of cells which had a better proliferative activity.RT-PCR demonstrated that expression of ERβmRNA was significantly suppressed in the cells treated with MOs, whereas in the cells treated with UMOs as control was not.MSP analysis showed that ERβgene promoter in the cells treated with MOs was hypermethylated compared with control. 6.Further study indicated that expressions of downstream targeted genes of estrogen signaling pathway were repressed simultaneously when ERβmRNA was significantly suppressed in the cells treated with MOs.What’s more,the activity of epigenetic enzymatic spectrum genes(Dnmt1,Dnmt3a,Dnmt3b and HMT) has changed at the same time.Conclusions:The present results suggested that except for the classical signaling pathway via estrogen reaction element,aging hypermethylation of ERβgene promoter can also remarkably mediate epigenetic regulation on its downstream targeted genes and consequently result in turbulence of this signaling pathway,which may benefit the effort to seek for the pathogenesis of the female ascending colon caner.Transcriptional inactivation of ERβgene in colonic epithelium cells may be mediated by promoter hypermethylation of ERβgene.Aging hypermethylation of ERβand its modulation on downstream targeted genes may play a significant role in carcinogenesis in colon cancer.Detection of the abnormal hypermethylation mode of ERβgene promoter will contribute to early diagnosis and prognosis evaluation of the female ascending coloh cancer.更多还原