溃疡性结肠炎microRNA的差异表达及MicroRNA-181a的功能研究

发布时间：2018-02-19 23:35

本文关键词： miR-181a 溃疡性结肠炎 TNF-α IκB　出处：《中国人民解放军医学院》2014年博士论文　论文类型：学位论文

【摘要】：背景溃疡性结肠炎（ulcerative colitis，UC）主要表现为粘液脓血便、反复发作性腹泻、腹痛，病变主要位于结肠的粘膜层和粘膜下层，是一种慢性非特异性的结肠炎症，属于炎症性肠病（inflammatory bowel disease, IBD）的一种。本病的确切病因不明，多种因素如遗传、感染、环境、免疫等在其发病过程中起到了重要的作用。肠道免疫系统调节异常，促炎细胞因子分泌增多可能是本病的主要发病机制，因此探讨肠道组织中炎症相关的信号转导过程有助于阐明UC的发病机制。 MicroRNA(miRNA)是一类长度为18-24个核苷酸的内源性非编码单链小分子RNA（nocoding RNA, ncRNA）。miRNA通过与靶基因3'非翻译区(3'UTR)的完全或不完全配对，降解靶基因mRNA或抑制其翻译负向调控基因表达，影响组织和细胞的功能。miRNA广泛参与生命过程中一系列重要进程，如肿瘤、炎症、细胞增殖、凋亡、组织器官的形成等。本研究旨在探讨UC中miRNA的差异表达以及miRNA-181a通过调控其靶基因TNF-α在UC发病机制中的作用，为阐明UC的发病机制提供新的理论依据。目的对课题小组前期的miRNA芯片结果进行分析，，选取在UC患者结肠粘膜中表达差异的5种miRNA：miR-181a、miR-181b、miR-182、miR-146a、miR-101，应用实时荧光定量RT-PCR方法进行验证。并探讨miR-181a与其靶基因TNF-α在UC患者肠道粘膜炎症发展中的作用及机制，为研究UC的发病机制提供新的切入点。方法 1.分析前期miRNA芯片结果筛选出5种在UC患者结肠粘膜组织中差异表达的miRNA:miR-181a、miR-181b、miR-182、miR-146a、miR-101，应用实时荧光定量RT-PCR法进一步进行验证。 2.选取miR-181a作为进一步的研究对象，利用生物信息学方法预测miR-181a可能的靶基因为TNF-α。Westernblot法检测UC患者和健康对照组结肠粘膜中TNF-α的表达变化。 3构建TNF-α3’UTR野生型及突变型载体，应用双荧光素酶报告基因实验检测miR-181a与TNF-α之间的靶向调控关系；过表达和抑制表达miR-181a进一步检测TNF-α在蛋白水平的变化。 4.过表达和抑制表达miR-181a检测TNF-α及下游IκB的表达变化。结果 1.实时荧光定量RT-PCR法检测发现在UC患者结肠粘膜中miR-146a、miR-101表达上调，miR-181a、miR-181b、miR-182表达下调。 2. Western blot法显示UC组结肠组织中TNF-α蛋白水平的明显高于健康对照组。 3.荧光素酶基因报告实验显示miR-181a可显著降低TNF-α3’UTR野生型组的荧光表达，而转染TNF-α3’UTR突变体组荧光表达升高；Western blot法显示过表达miR-181a组TNF-α蛋白水平的表达明显降低。 4Western blot法显示过表达miR-181a后TNF-α表达降低而IκB表达升高；抑制表达miR-181a后TNF-α表达升高及IκB表达降低。结论本课题对前期UC的miRNA芯片结果进行了分析，并应用实时荧光定量RT-PCR方法对其中5种miRNA进行检测，在UC患者结肠粘膜中miR-146a、miR-101表达上调；miR-181a、miR-181b、miR-182表达下调。证实TNF-α是miR-181a直接调控的靶基因，并可能通过调节NF-κB信号转导途径参与了UC结肠粘膜炎症的发生发展，为进一步深入研究UC的发病机制提供了新的途径。
[Abstract]:background
Ulcerative colitis (ulcerative colitis UC) is mainly mucusbloodandpus, recurrent diarrhea, abdominal pain, lesions are mainly located in the colon mucosa and submucosa, is a kind of chronic nonspecific colitis, belong to inflammatory bowel disease (inflammatory bowel, disease, IBD) is a kind of the exact cause of the disease. Unknown, many factors such as heredity, environment, infection, immunity plays an important role in the disease. The abnormal regulation of intestinal immune system, proinflammatory cytokine secretion may be the main pathogenesis of the disease, so the pathogenesis of inflammation related signal transduction in intestinal tissue contributes to the elucidation of UC.
MicroRNA (miRNA) is a class of endogenous non length of 18-24 nucleotides encoding small single stranded RNA (nocoding RNA ncRNA).MiRNA with target gene 3'untranslated region (3'UTR) of the complete or incomplete pairing, mRNA target gene degradation or inhibit translation negatively regulate gene expression, function and effect.MiRNA cells widely participate in the process of life in a series of important processes, such as tumor, inflammation, cell proliferation, apoptosis, tissue and organ formation. This study aimed to investigate the expression of UC in miRNA and miRNA-181a through the regulation of its target gene TNF- in the pathogenesis of UC and provide a new theoretical basis for elucidating the pathogenesis UC.
objective
The miRNA chip on the topic groupearlier period by analyzing the results of the selected expression of 5 miRNA:miR-181a, the differences in colonic mucosa of UC patients in miR-181b, miR-182, miR-146a, miR-101, by real-time quantitative RT-PCR. Verify and investigate in UC patients of intestinal mucosal inflammation in the development of the role and mechanism of miR-181a and its target gene TNF-, provide a new starting point for studying the pathogenesis of UC.
Method
1. analyze the previous miRNA chip results, and select 5 miRNA:miR-181a, miR-181b, miR-182, miR-146a and miR-101 expressed in colonic mucosa tissue of UC patients. The real-time fluorescent quantitative RT-PCR method is used for further validation.
2. select miR-181a as a further research object, and predict the possible target of miR-181a by bioinformatics. TNF- alpha.Westernblot method is used to detect the expression of TNF- alpha in colonic mucosa of UC patients and healthy controls.
3, we constructed the TNF- alpha 3 'UTR wild type and mutant vector. We used dual luciferase reporter gene assay to detect the target regulatory relationship between miR-181a and TNF- alpha, overexpressed and suppressed expression of miR-181a, and further detected the change of TNF- alpha at protein level.
4. over expression and inhibition of expression of miR-181a detected the expression of TNF- alpha and the expression of I kappa B downstream.
Result
1. the real-time fluorescence quantitative RT-PCR assay found that the expression of miR-146a and miR-101 in the colon mucosa of UC patients was up-regulated, and the expression of miR-181a, miR-181b, and miR-182 was down regulated.
The 2. Western blot method showed that the level of TNF- alpha protein in the colon tissue of UC group was significantly higher than that in the healthy control group.
3. luciferase gene reporter test showed that miR-181a significantly decreased the fluorescence expression of TNF- 3 'UTR wild type group, while the fluorescence expression of TNF- alpha 3' UTR mutant group increased. Western blot showed that the expression of TNF- alpha protein in miR-181a group was significantly lower than that in the miR-181a group.
4Western blot showed that the expression of TNF- alpha was decreased and the expression of I kappa B increased after overexpression of miR-181a, and the expression of TNF- A and the expression of I kappa B decreased after inhibiting the expression of miR-181a.
conclusion
The miRNA chip on the early results of UC were analyzed, and the 5 kinds of miRNA were detected by real-time fluorescence quantitative RT-PCR method, miR-146a in colonic mucosa of UC patients, expression of miR-181a, miR-181b, miR-101; expression of miR-182 alpha confirmed that TNF- was a target gene of miR-181a regulation, and may occur through development regulation of NF- B signal transduction pathway in UC colon inflammation, provides a new way for further research on the pathogenesis of UC.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574.62

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