發(fā)布時(shí)間：2018-06-12 22:21

  本文選題：TDG + Ras信號(hào)通路��； 參考：《浙江大學(xué)》2017年博士論文

【摘要】：背景胰腺癌是目前惡性程度最高的實(shí)體腫瘤,而且早期診斷十分困難,大部分患者初診時(shí)就處于疾病中晚期,無(wú)法手術(shù)切除,而且對(duì)放化療等內(nèi)科治療不敏感,因此總體5年生存率很低。近年來(lái),我國(guó)胰腺癌的發(fā)病率和死亡率仍在不斷上升,并已成為排名前十的最常見(jiàn)死亡相關(guān)癌癥之一。因此,明確胰腺癌等惡性腫瘤發(fā)病的生物學(xué)機(jī)制,為腫瘤診斷和治療提供新方法和新思路,無(wú)疑具有重要的科學(xué)價(jià)值和社會(huì)經(jīng)濟(jì)意義。作為第一個(gè)從人類(lèi)腫瘤細(xì)胞中分離出來(lái)的癌基因,Ras基因在胰腺癌中的突變發(fā)生率非常高。大量研究表明,胰腺癌的發(fā)生發(fā)展與Ras信號(hào)通路的異常激活密切相關(guān)。已有研究發(fā)現(xiàn),Ras信號(hào)通路異常激活導(dǎo)致的啟動(dòng)子甲基化失活是包括Fas在內(nèi)的多個(gè)抑癌基因在惡性腫瘤中功能缺失的原因。DNA主動(dòng)去甲基化是近年來(lái)表觀遺傳學(xué)領(lǐng)域的研究熱點(diǎn)。5-甲基胞嘧啶在TET雙加氧酶蛋白家族的催化下被不斷氧化形成5-羥甲基胞嘧啶、5-醛基胞嘧啶和5-羧基胞嘧啶,最終被胸腺嘧啶-DNA糖基化酶介導(dǎo)的堿基切除修復(fù)途徑還原為未修飾的胞嘧啶。同DNA甲基化一樣,DNA主動(dòng)去甲基化過(guò)程也具有重要的生物學(xué)意義,與細(xì)胞分化、個(gè)體發(fā)育以及惡性腫瘤等生理病理過(guò)程密切相關(guān)。因此,明確腫瘤發(fā)生過(guò)程中DNA主動(dòng)去甲基化相關(guān)酶的表達(dá)調(diào)控以及作用機(jī)制,將對(duì)闡明表觀遺傳調(diào)控機(jī)制,認(rèn)識(shí)腫瘤發(fā)病過(guò)程并探索新的腫瘤防治策略等具有重要意義。目的目前的DNA主動(dòng)去甲基化調(diào)控研究大多集中在TET蛋白家族上,針對(duì)TDG的研究相對(duì)較少。在本文中,我們將以Ras基因突變率較高的胰腺癌為主要研究對(duì)象,分析Ras信號(hào)通路對(duì)TDG的表達(dá)調(diào)控及其作用機(jī)制。我們將首先明確TDG是Ras信號(hào)通路的下游調(diào)控靶點(diǎn),并闡明其調(diào)控機(jī)制。然后分析其在胰腺癌細(xì)胞和病人組織樣本中的表達(dá)情況及其與胰腺癌的相關(guān)性。在此基礎(chǔ)上,探究TDG在胰腺癌發(fā)生發(fā)展中的功能及其作用機(jī)制。最后,通過(guò)分子生物學(xué)或生物化學(xué)手段改變TDG在細(xì)胞內(nèi)水平以驗(yàn)證其功能,為胰腺癌治療提供潛在的治療靶點(diǎn)。方法為研究Ras信號(hào)通路對(duì)TDG表達(dá)的影響,我們:①分別運(yùn)用免疫印跡和RT-PCR等方法比較轉(zhuǎn)染活化型Ras前后細(xì)胞內(nèi)TDG蛋白和mRNA的表達(dá)情況;②分別運(yùn)用免疫印跡和RT-PCR等方法比較siRNA敲減Ras前后攜帶k-Ras基因突變的胰腺癌細(xì)胞內(nèi)TDG蛋白和mRNA的表達(dá)情況;③利用免疫組化等技術(shù)檢測(cè)TDG在人胰腺癌組織標(biāo)本中的表達(dá)情況,分析TDG表達(dá)的臨床意義。為研究Ras信號(hào)通路調(diào)控TDG表達(dá)的機(jī)制,我們:①克隆TDG不同長(zhǎng)度啟動(dòng)子序列,采用熒光素酶報(bào)告基因系統(tǒng),確定TDG的活性啟動(dòng)子區(qū),分析Ras信號(hào)通路對(duì)該啟動(dòng)子活性的影響,并以此為基礎(chǔ),通過(guò)生物信息分析,篩選可結(jié)合該啟動(dòng)子的潛在轉(zhuǎn)錄因子。通過(guò)siRNA技術(shù)分別抑制這些潛在轉(zhuǎn)錄因子,發(fā)現(xiàn)抑癌基因ING4參與調(diào)控TDG表達(dá);②構(gòu)建ING4特異性結(jié)合位點(diǎn)的突變體,用熒光素酶報(bào)告基因系統(tǒng)檢驗(yàn)其活性,進(jìn)一步確定ING4對(duì)TDG的轉(zhuǎn)錄調(diào)控作用。轉(zhuǎn)染ING4的過(guò)表達(dá)載體,改變其表達(dá),觀察TDG的表達(dá)變化。通過(guò)染色質(zhì)免疫共沉淀技術(shù)驗(yàn)證TDG啟動(dòng)子與ING4的相互結(jié)合,并進(jìn)一步比較Ras信號(hào)通路活化前后二者間相互作用的改變;③在Ras轉(zhuǎn)化細(xì)胞中過(guò)表達(dá)或Ras敲減細(xì)胞中抑制ING4,比較TDG的表達(dá)情況,以驗(yàn)證ING4在Ras調(diào)控TDG表達(dá)過(guò)程中的作用。為驗(yàn)證TDG的功能及其作用機(jī)制,我們:①構(gòu)建了TDG的過(guò)表達(dá)載體,觀察過(guò)表達(dá)TDG前后腫瘤細(xì)胞的體外生長(zhǎng)能力和小鼠體內(nèi)成瘤能力;②用免疫印跡、流式細(xì)胞術(shù)、免疫組化等方法檢測(cè)細(xì)胞增殖標(biāo)記Ki-67、細(xì)胞凋亡標(biāo)記caspase-3剪切等在過(guò)表達(dá)TDG前后的腫瘤細(xì)胞或組織中的變化,以確定TDG對(duì)細(xì)胞增殖、凋亡等的影響,并由此發(fā)現(xiàn)死亡受體Fas是TDG的調(diào)控靶點(diǎn);③分別采用RT-PCR、免疫印跡或流式細(xì)胞檢測(cè)法,分析過(guò)表達(dá)TDG前后腫瘤細(xì)胞中Fas的表達(dá)變化,并比較Fas配體(FasL)對(duì)TDG表達(dá)前后細(xì)胞的凋亡誘導(dǎo)作用,以驗(yàn)證TDG對(duì)Fas表達(dá)的調(diào)控作用;④通過(guò)RT-PCR和免疫印跡方法驗(yàn)證TDG對(duì)Fas的調(diào)控作用,并確定TDG調(diào)控Fas表達(dá)的方式。通過(guò)染色質(zhì)免疫共沉淀等技術(shù),分析TDG與Fas啟動(dòng)子間的相互作用,比較TDG過(guò)表達(dá)前后,Fas基因的啟動(dòng)子甲基化、組蛋白修飾以及相關(guān)轉(zhuǎn)錄調(diào)控因子與Fas啟動(dòng)子間相互作用的改變;⑤在前述研究的基礎(chǔ)上,通過(guò)分子生物學(xué)(如基因敲除)或生物化學(xué)(如抑制劑)手段改變TDG或其調(diào)控因子在胰腺癌細(xì)胞中的表達(dá)或生物活性,初步檢驗(yàn)TDG或其調(diào)控因子作為腫瘤治療作用靶點(diǎn)的可能性。結(jié)果在本研究中,我們發(fā)現(xiàn)DNA主動(dòng)去甲基化過(guò)程關(guān)鍵酶TDG是Ras信號(hào)通路的下游調(diào)控基因,在Ras突變的胰腺癌細(xì)胞和胰腺癌組織中,TDG表達(dá)顯著下降。抑癌基因ING4通過(guò)與TDG啟動(dòng)子的特異性結(jié)合促進(jìn)TDG轉(zhuǎn)錄表達(dá),Ras信號(hào)通路以m-Calpain依賴(lài)的方式促進(jìn)ING4蛋白的降解,間接抑制TDG的轉(zhuǎn)錄。TDG能夠結(jié)合到死亡受體Fas基因的啟動(dòng)子區(qū)并招募組蛋白去甲基化酶JMJD3使組蛋白H3K27Me3去甲基化從而激活Fas的表達(dá),在體內(nèi)外誘導(dǎo)腫瘤細(xì)胞發(fā)生凋亡。Calpain抑制劑能夠提高ING4的蛋白穩(wěn)定性,使TDG、Fas的表達(dá)上調(diào),從而提高腫瘤細(xì)胞的凋亡敏感性。結(jié)論通過(guò)以上研究,我們明確了Ras對(duì)TDG的表達(dá)調(diào)控作用及其機(jī)制,闡明了TDG的腫瘤抑制作用及其機(jī)制,并在體內(nèi)外初步驗(yàn)證了 Calpain抑制劑作為一種潛在的胰腺癌靶向治療藥物的抗腫瘤效果,為胰腺癌診治提供了新的作用靶點(diǎn)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.9

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本文編號(hào)：2011284