胸腺嘧啶-DNA糖基化酶TDG在胰腺癌中的表达调控及作用机制研究

发布时间：2018-06-12 22:21

本文选题：TDG + Ras信号通路　；参考：《浙江大学》2017年博士论文

【摘要】：背景胰腺癌是目前恶性程度最高的实体肿瘤,而且早期诊断十分困难,大部分患者初诊时就处于疾病中晚期,无法手术切除,而且对放化疗等内科治疗不敏感,因此总体5年生存率很低。近年来,我国胰腺癌的发病率和死亡率仍在不断上升,并已成为排名前十的最常见死亡相关癌症之一。因此,明确胰腺癌等恶性肿瘤发病的生物学机制,为肿瘤诊断和治疗提供新方法和新思路,无疑具有重要的科学价值和社会经济意义。作为第一个从人类肿瘤细胞中分离出来的癌基因,Ras基因在胰腺癌中的突变发生率非常高。大量研究表明,胰腺癌的发生发展与Ras信号通路的异常激活密切相关。已有研究发现,Ras信号通路异常激活导致的启动子甲基化失活是包括Fas在内的多个抑癌基因在恶性肿瘤中功能缺失的原因。DNA主动去甲基化是近年来表观遗传学领域的研究热点。5-甲基胞嘧啶在TET双加氧酶蛋白家族的催化下被不断氧化形成5-羟甲基胞嘧啶、5-醛基胞嘧啶和5-羧基胞嘧啶,最终被胸腺嘧啶-DNA糖基化酶介导的碱基切除修复途径还原为未修饰的胞嘧啶。同DNA甲基化一样,DNA主动去甲基化过程也具有重要的生物学意义,与细胞分化、个体发育以及恶性肿瘤等生理病理过程密切相关。因此,明确肿瘤发生过程中DNA主动去甲基化相关酶的表达调控以及作用机制,将对阐明表观遗传调控机制,认识肿瘤发病过程并探索新的肿瘤防治策略等具有重要意义。目的目前的DNA主动去甲基化调控研究大多集中在TET蛋白家族上,针对TDG的研究相对较少。在本文中,我们将以Ras基因突变率较高的胰腺癌为主要研究对象,分析Ras信号通路对TDG的表达调控及其作用机制。我们将首先明确TDG是Ras信号通路的下游调控靶点,并阐明其调控机制。然后分析其在胰腺癌细胞和病人组织样本中的表达情况及其与胰腺癌的相关性。在此基础上,探究TDG在胰腺癌发生发展中的功能及其作用机制。最后,通过分子生物学或生物化学手段改变TDG在细胞内水平以验证其功能,为胰腺癌治疗提供潜在的治疗靶点。方法为研究Ras信号通路对TDG表达的影响,我们:①分别运用免疫印迹和RT-PCR等方法比较转染活化型Ras前后细胞内TDG蛋白和mRNA的表达情况;②分别运用免疫印迹和RT-PCR等方法比较siRNA敲减Ras前后携带k-Ras基因突变的胰腺癌细胞内TDG蛋白和mRNA的表达情况;③利用免疫组化等技术检测TDG在人胰腺癌组织标本中的表达情况,分析TDG表达的临床意义。为研究Ras信号通路调控TDG表达的机制,我们:①克隆TDG不同长度启动子序列,采用荧光素酶报告基因系统,确定TDG的活性启动子区,分析Ras信号通路对该启动子活性的影响,并以此为基础,通过生物信息分析,筛选可结合该启动子的潜在转录因子。通过siRNA技术分别抑制这些潜在转录因子,发现抑癌基因ING4参与调控TDG表达;②构建ING4特异性结合位点的突变体,用荧光素酶报告基因系统检验其活性,进一步确定ING4对TDG的转录调控作用。转染ING4的过表达载体,改变其表达,观察TDG的表达变化。通过染色质免疫共沉淀技术验证TDG启动子与ING4的相互结合,并进一步比较Ras信号通路活化前后二者间相互作用的改变;③在Ras转化细胞中过表达或Ras敲减细胞中抑制ING4,比较TDG的表达情况,以验证ING4在Ras调控TDG表达过程中的作用。为验证TDG的功能及其作用机制,我们:①构建了TDG的过表达载体,观察过表达TDG前后肿瘤细胞的体外生长能力和小鼠体内成瘤能力;②用免疫印迹、流式细胞术、免疫组化等方法检测细胞增殖标记Ki-67、细胞凋亡标记caspase-3剪切等在过表达TDG前后的肿瘤细胞或组织中的变化,以确定TDG对细胞增殖、凋亡等的影响,并由此发现死亡受体Fas是TDG的调控靶点;③分别采用RT-PCR、免疫印迹或流式细胞检测法,分析过表达TDG前后肿瘤细胞中Fas的表达变化,并比较Fas配体(FasL)对TDG表达前后细胞的凋亡诱导作用,以验证TDG对Fas表达的调控作用;④通过RT-PCR和免疫印迹方法验证TDG对Fas的调控作用,并确定TDG调控Fas表达的方式。通过染色质免疫共沉淀等技术,分析TDG与Fas启动子间的相互作用,比较TDG过表达前后,Fas基因的启动子甲基化、组蛋白修饰以及相关转录调控因子与Fas启动子间相互作用的改变;⑤在前述研究的基础上,通过分子生物学(如基因敲除)或生物化学(如抑制剂)手段改变TDG或其调控因子在胰腺癌细胞中的表达或生物活性,初步检验TDG或其调控因子作为肿瘤治疗作用靶点的可能性。结果在本研究中,我们发现DNA主动去甲基化过程关键酶TDG是Ras信号通路的下游调控基因,在Ras突变的胰腺癌细胞和胰腺癌组织中,TDG表达显著下降。抑癌基因ING4通过与TDG启动子的特异性结合促进TDG转录表达,Ras信号通路以m-Calpain依赖的方式促进ING4蛋白的降解,间接抑制TDG的转录。TDG能够结合到死亡受体Fas基因的启动子区并招募组蛋白去甲基化酶JMJD3使组蛋白H3K27Me3去甲基化从而激活Fas的表达,在体内外诱导肿瘤细胞发生凋亡。Calpain抑制剂能够提高ING4的蛋白稳定性,使TDG、Fas的表达上调,从而提高肿瘤细胞的凋亡敏感性。结论通过以上研究,我们明确了Ras对TDG的表达调控作用及其机制,阐明了TDG的肿瘤抑制作用及其机制,并在体内外初步验证了 Calpain抑制剂作为一种潜在的胰腺癌靶向治疗药物的抗肿瘤效果,为胰腺癌诊治提供了新的作用靶点。
[Abstract]:Background pancreatic cancer is the most malignant solid tumor at present, and early diagnosis is very difficult. Most patients are in the middle and late stages of the disease, unable to be excised, and are insensitive to chemotherapy and chemotherapy, so the overall 5 year survival rate is very low. In recent years, the incidence and mortality of pancreatic cancer in China are still rising. It has become one of the most common death related cancers in the top ten. Therefore, it is of great scientific value and socioeconomic significance to clarify the biological mechanism of the pathogenesis of pancreatic cancer and to provide new methods and new ideas for the diagnosis and treatment of cancer. As the first oncogene isolated from human tumor cells, Ras The mutation rate in the pancreatic cancer is very high. A large number of studies have shown that the development of pancreatic cancer is closely related to the abnormal activation of the Ras signaling pathway. It has been found that the deactivation of the promoter induced by abnormal activation of the Ras signaling pathway is the cause of the function loss of multiple tumor suppressor genes, including Fas,.DNA, in the malignant tumor. Active demethylation is a hot spot in epigenetics in recent years,.5- methyl cytosine is continuously oxidized to 5- hydroxymethyl cytosine, 5- aldehyde based cytosine and 5- carboxylic cytosine under the catalysis of TET dioxygenase family, and eventually reduced to unmodified by thymine -DNA glycosylase. Cytosine. Like DNA methylation, DNA active demethylation also has important biological significance, which is closely related to the physiological and pathological processes such as cell differentiation, ontogenesis and malignant tumor. Therefore, the regulation of the expression of DNA active demethylation related enzymes and the mechanism of action in the process of tumor occurrence will clarify epigenetic regulation. Control mechanism, understanding the process of tumor onset and exploring new tumor prevention strategies are of great significance. The research of active demethylation of DNA is mostly focused on the TET protein family, and the research on TDG is relatively small. In this paper, we will take the pancreatic cancer with high Ras mutation rate as the main research object and analyze the Ras letter. The regulation of TDG expression and its mechanism of action. We will first make clear that TDG is the downstream regulatory target of Ras signaling and elucidates its regulatory mechanism, and then analyzes its expression in pancreatic cancer cells and patients' tissue samples and the correlation with pancreatic cancer. On this basis, the work of TDG in the development of pancreatic cancer is explored. Ability and its mechanism of action. Finally, using molecular or biochemical means to change the intracellular level of TDG to verify its function and provide potential therapeutic targets for the treatment of pancreatic cancer. Methods to study the effect of Ras signaling pathway on the expression of TDG, we: 1. The immunological trace and RT-PCR were used to compare the transfection of activated Ras before and after transfection. The expression of TDG protein and mRNA in cells, and the expression of TDG protein and mRNA in pancreatic cancer cells with k-Ras gene mutation before and after siRNA knockout Ras were compared by immunoblotting and RT-PCR, respectively. Thirdly, the expression of TDG in human pancreatic cancer tissue was detected by immunohistochemical technique and the clinical expression of TDG was analyzed. In order to study the mechanism of regulating TDG expression in Ras signaling pathway, we: (1) cloning TDG of different length promoter sequences, using luciferase reporter gene system, determining the active promoter region of TDG, and analyzing the effect of Ras signaling pathway on the activity of the promoter, and on this basis, through bioinformatics analysis, screening can be used to combine the promoter. Potential transcription factors. The potential transcriptional factors were suppressed by siRNA technology, and the tumor suppressor gene ING4 was found to be involved in the regulation of TDG expression; (2) the mutant of the ING4 specific binding site was constructed, the activity was detected by the luciferase reporter gene system, and the transcriptional regulation of ING4 to TDG was further determined. The over expression vector of ING4 was transfected to change its expression vector. Expression and observation of expression changes of TDG. The interaction of TDG promoter and ING4 was verified by chromatin immunoprecipitation technique, and the interaction between the two groups was further compared before and after the activation of Ras signaling pathway. (3) ING4 was suppressed in Ras transformed cells or Ras knockout cells, and the expression of TDG was compared in order to verify ING4 in Ras. In order to verify the function and mechanism of the expression of TDG, in order to verify the function and mechanism of TDG, we have constructed the overexpression vector of TDG, observed the growth ability of the tumor cells in vitro and the tumor formation ability of the tumor cells before and after the expression of TDG; (2) the cell proliferation marker Ki-67 was detected by immunoblotting, flow cytometry and immunohistochemistry. The changes in the tumor cells or tissues before and after the overexpression of TDG were recorded by Caspase-3, in order to determine the effect of TDG on cell proliferation and apoptosis, and to find that the death receptor Fas was a regulatory target of TDG; (3) the expression of Fas in the tumor cells before and after the expression of TDG was analyzed by RT-PCR, immunoblotting or flow cytometry, and To compare the apoptosis induction of Fas ligand (FasL) before and after TDG expression in order to verify the regulation of TDG on Fas expression; (4) to verify the regulation of Fas by RT-PCR and immunoblotting, and to determine the way TDG regulates the expression of Fas. The interaction between TDG and Fas promoter is analyzed by chromatin immunoprecipitation. The promoter methylation of the Fas gene, histone modification, and changes in the interaction between the related transcriptional regulators and Fas promoters before and after TDG overexpression; 5. On the basis of the previous study, the expression of TDG or its regulators in pancreatic cancer cells is altered by molecular biology (such as gene knockout) or biochemical (such as inhibitors). In this study, we found that the key enzyme of the DNA active demethylation process, TDG, is a downstream regulatory gene of the Ras signaling pathway in this study. In this study, we found that the expression of TDG in the Ras mutant pancreatic and pancreatic cancer tissues was significantly reduced. The tumor suppressor gene ING4 was expressed in ING4. The specific binding with the TDG promoter promotes TDG transcriptional expression. The Ras signaling pathway promotes the degradation of ING4 protein in a m-Calpain dependent manner, and indirectly inhibits the TDG transcriptional.TDG to bind to the promoter region of the death receptor Fas gene and recruit the histone demethylation JMJD3 to demethylation of histone H3K27Me3 so as to activate Fas expression. In vivo and in vivo, inducing tumor cell apoptosis.Calpain inhibitors can improve the protein stability of ING4, up the expression of TDG, Fas, and increase the apoptosis sensitivity of tumor cells. Conclusion through the above study, we clarify the regulatory role of Ras on the expression of TDG and its mechanism, elucidate the inhibitory effect of TDG and its mechanism, and The anti tumor effect of Calpain inhibitor as a potential therapeutic target for pancreatic cancer is preliminarily verified in vitro and in vivo, which provides a new target for the diagnosis and treatment of pancreatic cancer.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.9

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