本文選題：5-羥甲基胞嘧啶　切入點(diǎn)：原發(fā)性肝細(xì)胞性肝癌　出處：《武漢大學(xué)》2015年博士論文

【摘要】：目的：肝臟是糖脂代謝的重要器官,2型糖尿病(type 2 diabetes mellitus, T2DM)是人類最常見的糖脂代謝紊亂性疾病。表觀遺傳學(xué)修飾是遺傳因素、環(huán)境因素和生活方式綜合作用的結(jié)果。核酸修飾紊亂與肝癌和T2DM的發(fā)生發(fā)展密切相關(guān),5-甲基胞嘧啶(5-methylcytosine,5mC)是DNA甲基化的最常見類型,而DNA去甲基化中間產(chǎn)物5-羥甲基胞嘧啶(5-hydroxymethylcytosine,5hmC)的發(fā)現(xiàn)為進(jìn)一步明確DNA表觀遺傳學(xué)在癌癥中的作用提供了重要依據(jù)。既往研究證實(shí)肥胖相關(guān)(fat mass- and obesity-associated, FTO)蛋白的高表達(dá)與T2DM及肥胖有一定的關(guān)聯(lián),最近研究發(fā)現(xiàn)它為RNA去甲基化酶,但它在疾病中的表觀遺傳調(diào)節(jié)作用目前尚無報(bào)道。本研究第一部分以原發(fā)性肝細(xì)胞性肝癌(hepatocellular carcinoma, HCC)作為研究對(duì)象,探索DNA去甲基化中間產(chǎn)物5hmC在HCC發(fā)生中的作用；第二部分和第三部分以T2DM作為研究對(duì)象,揭示RNA甲基化紊亂在該疾病中的作用。期望找到HCC和T2DM的早期診斷指標(biāo),為尋求該疾病的新的治療手段奠定基礎(chǔ)。材料與方法：在武漢大學(xué)中南醫(yī)院收集HCC患者肝臟腫瘤福爾馬林固定石蠟包埋癌組織及癌旁組織標(biāo)本用于DNA表觀遺傳學(xué)研究；收集T2DM患者及性別年齡匹配的健康對(duì)照者外周全血標(biāo)本,并構(gòu)建鏈脲佐菌素(streptozotocin, STZ)處理的糖尿病大鼠和正常Sprague Dawley大鼠模型用于RNA表觀遺傳學(xué)研究。采用新創(chuàng)建的親水毛細(xì)管色譜-飛行時(shí)間質(zhì)譜聯(lián)用技術(shù)(capillary hydrophilic-interaction liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry, cHILIC-ESI-qTOF-MS)檢測(cè)HCC患者癌組織和癌旁組織全基因組DNA中5mC和5hmC的含量；采用課題組創(chuàng)立的液相色譜-質(zhì)譜聯(lián)用技術(shù)(liquid chromatography-electrospray ionization-tandem mass spectrometry, LC-ESI-MS/MS)檢測(cè)T2DM患者、健康對(duì)照以及大鼠模型外周血全基因組總RNA中的N6-甲基腺嘌呤(N6-methyladenosine, m6A)的含量；采用實(shí)時(shí)熒光定量PCR (real-time quantitative PCR,RT-qPCR)技術(shù)分別檢測(cè)HCC患者肝臟腫瘤組織中線粒體DNA(mitochondrial DNA, mtDNA)的含量和T2DM患者、健康對(duì)照以及大鼠模型外周全血中RNA去甲基化酶-FTO、α-酮戊二酸-依賴性加雙氧酶alkB同源蛋白5 (alkB homolog5, ALKBH5)和RNA甲基轉(zhuǎn)移酶-甲基轉(zhuǎn)移酶3(methyltransferaselike 3, METTL3)、甲基轉(zhuǎn)移酶14 (methyltransferase like 14, METTL14)、Wilms腫瘤相關(guān)蛋白(Wilms tumor 1 associated protein, WTAP)的mRNA表達(dá)水平；高分辨熔解曲線(high-resolution melting, HRM)法和DNA測(cè)序法檢測(cè)FTO基因的四種常見的單個(gè)核苷酸多態(tài)性(single-nucleotide polymorphisms, SNPs);并采用SPSS軟件分析實(shí)驗(yàn)結(jié)果。結(jié)論：DNA表觀遺傳學(xué)研究結(jié)果證明,HCC患者癌組織中5hmC含量顯著性低于對(duì)應(yīng)的癌旁組織,且5hmC含量與腫瘤分期和腫瘤大小密切相關(guān),揭示了5hmC的缺失在HCC的表觀遺傳學(xué)調(diào)節(jié)中的重要作用,它可能作為潛在的HCC的早期檢測(cè)生物指標(biāo)；而HCC腫瘤組織中的mtDNA含量與5mC含量明顯負(fù)相關(guān),與5hmC相關(guān)性不明顯,mtDNA、5mC和5hmC在HCC腫瘤組織基因組之間的關(guān)聯(lián)為HCC的發(fā)病機(jī)制研究提供了新思路。RNA表觀遺傳學(xué)研究證明,T2DM患者和糖尿病模型大鼠外周血中RNA去甲基化酶FTO基因的mRNA表達(dá)水平較正常對(duì)照組顯著升高；FTO基因的1mRNA表達(dá)水平與m6A的含量和T2DM的發(fā)病風(fēng)險(xiǎn)均密切相關(guān)。說明T2DM患者FTO基因的mRNA表達(dá)水平的升高可能是受到了降低的m6A的調(diào)節(jié)作用,它們也可能與T2DM的并發(fā)癥密切相關(guān)；升高的FTO基因mRNA表達(dá)水平可能作為輔助診斷T2DM的一個(gè)潛在的生物學(xué)標(biāo)記。此外,T2DM患者M(jìn)ETTL3、METTL14、WTAP基因的mRNA表達(dá)水平較健康對(duì)照組明顯升高,在糖尿病模型鼠中的也得到相似結(jié)論；METTL14基因mRNA表達(dá)水平與m6A含量負(fù)相關(guān)。且METTL3、METTL14和WTAP的]mRNA表達(dá)水平與FTO和ALKBH5的mRNA表達(dá)水平均正相關(guān)。說明RNA甲基轉(zhuǎn)移酶復(fù)合物與RNA去甲基化酶在T2DM的RNA甲基化調(diào)節(jié)中起到相輔相成的作用,METTL14可能在甲基轉(zhuǎn)移酶復(fù)合物中起到了核心作用,晶體結(jié)構(gòu)的研究將有助于進(jìn)一步證實(shí)其重要作用。
[Abstract]:Objective: the liver is an important organ in glucose and lipid metabolism in type 2 diabetes (type 2, diabetes mellitus, T2DM) is the most common disease of lipid metabolic disorders in humans. Epigenetics is genetic factors, the comprehensive effect of environmental factors and lifestyle. Closely related to the occurrence and development of hepatocellular carcinoma and nucleic acid modified disorders and T2DM, 5- 5-methylcytosine (5-methylcytosine, 5mC) is the most common type of DNA methylation and demethylation of DNA intermediate 5- hydroxymethylcytosine (5-hydroxymethylcytosine, 5hmC) was found to further define the DNA epigenetic provides an important basis for the study on the role of cancer. Previous studies have shown that obesity related mass- (fat and obesity-associated, FTO) high expression protein was associated with T2DM and obesity, a recent study found it demethylase RNA, but it is in the disease in the epigenetic regulation it is No reports. The first part of this study in primary hepatocellular carcinoma (hepatocellular, carcinoma, HCC) as the research object, to explore the demethylation of DNA intermediate 5hmC role in HCC formation; the second part and the third part to T2DM as the research object, to reveal the role of RNA methylation in the disorder disease. Early the diagnosis index expected to find HCC and T2DM, to lay the foundation for a new treatment for this disease. Materials and methods: HCC patients with liver tumors in formalin fixed paraffin embedded cancer tissue and adjacent tissue specimens for DNA epigenetic research in Zhongnan Hospital of Wuhan University; collect T2DM patients and age and gender matched healthy controls the peripheral blood samples, and the construction of streptozotocin (streptozotocin, STZ) treated diabetic rats and normal Sprague rat model of Dawley RNA for epigenetic research. The newly created hydrophilic capillary chromatography time of flight mass spectrometry (capillary hydrophilic-interaction liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry, cHILIC-ESI-qTOF-MS) were detected in HCC cancer tissue and paracancerous tissue 5mC and 5hmC genome in DNA; the research group founded by liquid chromatography-mass spectrometry (liquid chromatography-electrospray ionization-tandem mass spectrometry LC-ESI-MS/MS), detection of T2DM patients and healthy control rat model of peripheral blood genome total RNA N6- methyladenine (N6-methyladenosine, m6A) content; by using real-time quantitative PCR (real-time quantitative PCR, RT-qPCR) were detected in mitochondrial DNA in patients with HCC liver tumor tissues (mitochondrial DNA, mtDNA) content health and T2DM patients. RNA demethylase -FTO control and rat model of peripheral blood, alpha ketoglutarate dependent dioxygenase alkB homolog 5 (alkB homolog5, ALKBH5) and RNA methyltransferase - methyltransferase 3 (methyltransferaselike 3, METTL3), methyltransferase 14 (methyltransferase like 14, METTL14 Wilms), tumor associated protein (Wilms tumor 1 associated protein, WTAP) the expression level of mRNA; high resolution melting curve (high-resolution melting, HRM) four common single nucleotide polymorphism detection of FTO gene and DNA sequencing (single-nucleotide polymorphisms, SNPs); and using SPSS software to analyze the experimental results. Conclusion DNA: epigenetic research results show that the content of 5hmC in cancer tissues of HCC patients was significantly lower than that in paracancerous tissues, and the content of 5hmC and tumor stage and tumor size are closely related, reveals the absence of 5hmC The concept of an important role in the regulation of genetics in the HCC table, it could be detected as early as the biological indicators of potential HCC; while the content of mtDNA and the content of 5mC and HCC in tumor tissue was negatively correlated, the correlation between 5hmC and mtDNA is not obvious, and provides new ideas for epigenetic research proved that.RNA 5mC and 5hmC in the association between HCC the tumor tissue genome to study the pathogenesis of HCC in patients with T2DM and diabetic rats in the peripheral blood of RNA demethylation of FTO gene expression level of mRNA was significantly higher than normal control group; the risk content and T2DM FTO gene expression level of 1mRNA and m6A were closely related to that of FTO gene in patients with T2DM. The expression level of mRNA may be increased by regulating effect of reduced m6A, they may also be associated with complications of T2DM are closely related; the expression level of FTO gene increased mRNA may be used as auxiliary diagnostic T2DM A potential biological marker. In addition, with T2DM METTL3, METTL14, WTAP gene expression level of mRNA compared with the healthy control group significantly increased in the diabetic rat model which has the similar conclusion; METTL14 gene expression of mRNA is negatively correlated with the m6A content. And the level of METTL3, the expression level of METTL14 and WTAP and]mRNA expression level FTO and ALKBH5 mRNA were positive correlation. The results showed that RNA methyltransferase complexes with RNA demethylase plays a complementary role in the T2DM of RNA methylation in the regulation of METTL14 may transfer the enzyme complex in methyl has played a central role in the study of crystal structure, will help to further confirm the important role.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R587.1;R735.7

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