核酸修饰紊乱与肝癌和2型糖尿病的相关性及机制研究

发布时间：2018-04-03 20:28

本文选题：5-羟甲基胞嘧啶　切入点：原发性肝细胞性肝癌　出处：《武汉大学》2015年博士论文

【摘要】：目的：肝脏是糖脂代谢的重要器官,2型糖尿病(type 2 diabetes mellitus, T2DM)是人类最常见的糖脂代谢紊乱性疾病。表观遗传学修饰是遗传因素、环境因素和生活方式综合作用的结果。核酸修饰紊乱与肝癌和T2DM的发生发展密切相关,5-甲基胞嘧啶(5-methylcytosine,5mC)是DNA甲基化的最常见类型,而DNA去甲基化中间产物5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5hmC)的发现为进一步明确DNA表观遗传学在癌症中的作用提供了重要依据。既往研究证实肥胖相关(fat mass- and obesity-associated, FTO)蛋白的高表达与T2DM及肥胖有一定的关联,最近研究发现它为RNA去甲基化酶,但它在疾病中的表观遗传调节作用目前尚无报道。本研究第一部分以原发性肝细胞性肝癌(hepatocellular carcinoma, HCC)作为研究对象,探索DNA去甲基化中间产物5hmC在HCC发生中的作用；第二部分和第三部分以T2DM作为研究对象,揭示RNA甲基化紊乱在该疾病中的作用。期望找到HCC和T2DM的早期诊断指标,为寻求该疾病的新的治疗手段奠定基础。材料与方法：在武汉大学中南医院收集HCC患者肝脏肿瘤福尔马林固定石蜡包埋癌组织及癌旁组织标本用于DNA表观遗传学研究；收集T2DM患者及性别年龄匹配的健康对照者外周全血标本,并构建链脲佐菌素(streptozotocin, STZ)处理的糖尿病大鼠和正常Sprague Dawley大鼠模型用于RNA表观遗传学研究。采用新创建的亲水毛细管色谱-飞行时间质谱联用技术(capillary hydrophilic-interaction liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry, cHILIC-ESI-qTOF-MS)检测HCC患者癌组织和癌旁组织全基因组DNA中5mC和5hmC的含量；采用课题组创立的液相色谱-质谱联用技术(liquid chromatography-electrospray ionization-tandem mass spectrometry, LC-ESI-MS/MS)检测T2DM患者、健康对照以及大鼠模型外周血全基因组总RNA中的N6-甲基腺嘌呤(N6-methyladenosine, m6A)的含量；采用实时荧光定量PCR (real-time quantitative PCR,RT-qPCR)技术分别检测HCC患者肝脏肿瘤组织中线粒体DNA(mitochondrial DNA, mtDNA)的含量和T2DM患者、健康对照以及大鼠模型外周全血中RNA去甲基化酶-FTO、α-酮戊二酸-依赖性加双氧酶alkB同源蛋白5 (alkB homolog5, ALKBH5)和RNA甲基转移酶-甲基转移酶3(methyltransferaselike 3, METTL3)、甲基转移酶14 (methyltransferase like 14, METTL14)、Wilms肿瘤相关蛋白(Wilms tumor 1 associated protein, WTAP)的mRNA表达水平；高分辨熔解曲线(high-resolution melting, HRM)法和DNA测序法检测FTO基因的四种常见的单个核苷酸多态性(single-nucleotide polymorphisms, SNPs);并采用SPSS软件分析实验结果。结论：DNA表观遗传学研究结果证明,HCC患者癌组织中5hmC含量显著性低于对应的癌旁组织,且5hmC含量与肿瘤分期和肿瘤大小密切相关,揭示了5hmC的缺失在HCC的表观遗传学调节中的重要作用,它可能作为潜在的HCC的早期检测生物指标；而HCC肿瘤组织中的mtDNA含量与5mC含量明显负相关,与5hmC相关性不明显,mtDNA、5mC和5hmC在HCC肿瘤组织基因组之间的关联为HCC的发病机制研究提供了新思路。RNA表观遗传学研究证明,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表达水平较正常对照组显著升高；FTO基因的1mRNA表达水平与m6A的含量和T2DM的发病风险均密切相关。说明T2DM患者FTO基因的mRNA表达水平的升高可能是受到了降低的m6A的调节作用,它们也可能与T2DM的并发症密切相关；升高的FTO基因mRNA表达水平可能作为辅助诊断T2DM的一个潜在的生物学标记。此外,T2DM患者METTL3、METTL14、WTAP基因的mRNA表达水平较健康对照组明显升高,在糖尿病模型鼠中的也得到相似结论；METTL14基因mRNA表达水平与m6A含量负相关。且METTL3、METTL14和WTAP的]mRNA表达水平与FTO和ALKBH5的mRNA表达水平均正相关。说明RNA甲基转移酶复合物与RNA去甲基化酶在T2DM的RNA甲基化调节中起到相辅相成的作用,METTL14可能在甲基转移酶复合物中起到了核心作用,晶体结构的研究将有助于进一步证实其重要作用。
[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.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.1;R735.7

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