子宫腺肌病在位内膜长链非编码RNA和信使RNA差异表达研究

发布时间：2018-10-26 19:09

【摘要】：研究背景及目的子宫腺肌病是女性常见疾病,主要临床表现为不同程度的继发性进行性痛经、异常子宫出血,可能导致不孕不育和早期流产等,严重影响女性的身心健康和生活质量。至今为止其发病机制仍不清楚。目前关于其发病机制存在多种学说,最广为接受的学说认为子宫内膜内陷于子宫肌层并异位增殖导致该病的发生。研究发现,该病的在位内膜存在多种异常,涉及遗传、激素、免疫和代谢等多个方面。但是编码基因相关的研究较为零散,通过组学的方法系统筛选该病在位内膜编码基因的改变有助于揭示该病的发生机制。除了编码基因的改变以外,也存在表观遗传的异常变化。lncRNAs作为功能调节元件在基因表达调控中发挥重要的作用,并参与多种重要信号通路的调节。其异常表达与多种良恶性疾病相关。lncRNAs在子宫腺肌病发生发展中的作用尚无研究报道。本研究的目的为：1. 构建子宫腺肌病在位内膜与对照内膜lncRNAs和nRNAs的差异表达谱；2. 对lncRNA和mRNA的功能进行生物学信息分析,以探讨其可能的调控机制并得出潜在的核心调节基因；3. 对部分潜在核心调节基因的表达水平进行检测,为后续功能实验提供研究基础。研究方法1. 利用基因芯片技术检测子宫腺肌病在位内膜和对照内膜组织中lncRNAs和mRNAs的表达,构建lncRNAs和mRNAs差异表达谱(其中两组的样本量分别为4例)。2. 利用实时荧光定量PCR技术验证芯片结果中部分差异表达IncRNAs(共6个),以进一步验证基因芯片实验结果。3. 利用生物信息学手段对差异表达的lncRNAs和mRNAs进行分析。3.1 GO分析：对芯片结果中差异mRNAs进行显著性功能分析,得到差异基因参与的具有显著性、低误判率、靶向性的功能。3.2 Pathway分析：将差异nRNAs进行Pathway显著性分析,得到差异基因参与的具有显著性、低误判率、靶向性的Pathway。3.3构建共表达网络：通过实验组与对照组比较得到的差异mRNAs、差异lncRNAs在芯片中的实测表达值,分别构建实验组与对照组的共表达网络,通过比较共表达网络上的差异来定位两组样本中的潜在核心调节基因。4. 利用实时荧光定量PCR和免疫组化法检测部分潜在核心调节基因——TLN1和CCND2在子宫腺肌病在位内膜中的表达。研究结果1. 子宫腺肌病在位内膜与对照内膜相比,存在165个差异表达lncRNAs和61 2个差异表达mRNAs。在差异表达的lncRNAs中有117个lncRNAs表达下调和48个lncRNAs表达上调。其中下调最明显的lncRNA为n333955(差异倍数为：0.0032),上调最明显的lncRNA为n342839(差异倍数为4.13)。在差异表达的mRNAs中,下调的nRNAs,总数为414个,上调的mRNAs总数198个。其中下调最明显的mRNA为HBA1(差异倍数为：0.02),上调最明显的mRNA为THBS2(差异倍数为5.14)。2. 选择6个lncRNAs进行实时荧光定量PCR验证,结果显示n333955、n337373、n338909这3个lncRNAs在子宫腺肌病在位内膜组织中表达水平下降,而n341651、n342794、n387706表达水平升高,差异均具有统计学意义,与芯片检测的表达趋势相符。3. 生物学信息分析结果：3.1通过显著靶向性GO分析,得出上调差异基因参与的显著性功能共352项,包括：细胞外基质组成、内皮细胞分化、细胞内信号转导、细胞迁移的正调控、血管生成等。下调差异基因参与的显著性功能共283项,包括：基因表达、DNA复制、细胞分裂、有丝分裂细胞周期的G1/S期过渡、免疫反应等。3.2通过显著性Pathway分析,得到上调差异基因参与的显著性信号转导通路共40项,包括MAPK信号通路、局部粘附、粘附连接、细胞外基质受体相互作用、PI3K-Akt信号通路等。下调差异基因参与的显著性信号转导通路共39项,包括：DNA复制、细胞周期、原发性免疫缺陷、细胞因子、细胞因子受体相互作用等。3.3分别构建了两组样本的共表达网络,两张共表达网络的结构存在明显差别。根据基因在两个网络中共表达地位的变化情况,筛选出对样本差异起重要作用的关键mRNAs/lncRNAs,包括TLN1、n342794、MYBL1、CCND2、 ENST00000406939、n338909等4. 实时荧光定量PCR和免疫组化结果显示,TLN1基因和蛋白在子宫腺肌病在位内膜组织中的表达明显高于对照内膜组织；CCND2基因和蛋白在子宫腺肌病在位内膜组织中的表达明显低于对照内膜组织,差异均具有统计学意义。研究结论1. 首次通过芯片检测成功构建子宫腺肌病在位内膜与对照内膜lncRNAs和mRNAs的差异表达谱。2. 实时荧光定量PCR结果与芯片结果有很好的一致性,进一步验证芯片结果的可信度。3. 通过生物信息学分析可初步探讨差异mRNAs和lncRNAs在子宫腺肌病发生过程中可能的调控作用。4. 潜在核心调节基因TLN1和CCND2基因和蛋白在子宫腺肌病在位内膜组织中表达异常。
[Abstract]:The study background and objective uterine adenomyosis are female urinary tract diseases. The main clinical manifestations include secondary progressive dysmenorrhea, abnormal uterine bleeding, infertility and early abortion, which seriously affect women's physical and mental health and quality of life. Until now its pathogenesis remains unclear. There are many theories about the pathogenesis of the disease, and the most widely accepted theory holds that the endometriosis is trapped in the uterine muscle layer and ectopic proliferation leads to the occurrence of the disease. It has been found that there are a variety of abnormalities in the endometrium of the disease, which involve many aspects such as heredity, hormone, immunity and metabolism. But the research on coding gene is fragmented, and the method of group learning can screen the change of the coding gene of the disease in order to reveal the pathogenesis of the disease. In addition to the change in the coding gene, there is an abnormal change in apparent genetic. It plays an important role in the regulation of gene expression as a function regulating element, and participates in the regulation of various important signal paths. Its abnormal expression is associated with various benign and malignant diseases. There is no study in the development of adenomyosis. The purpose of this study is: 1. To construct a differential expression profile between the endometrium of the adenomyosis and the endometrium of the control endometrium; 2. Biological information analysis was carried out on the functions of cRNA and mRNA to investigate possible regulatory mechanisms and to develop potential core regulatory genes; 3. The expression level of some potential core regulatory genes was tested to provide a basis for follow-up function experiments. Study Method 1. Gene chip technique was used to detect the expression of Jurkat and mCD44v6 in endometrial and control endometrial tissues, and the differential expression profiles were constructed (the sample sizes of the two groups were 4 cases, respectively). The results of gene chip experiment were verified by using real-time fluorescence quantitative polymerase chain reaction (PCR) technique to verify partial differential expression in the chip. 3. 1GO analysis: Significant sexual function analysis was performed on the differences in the results of the chip, and the differentially expressed genes were found to have significance, low misjudgment rate and targeting function. A co-expression network with significance, low misjudgment rate and targeting property of differential gene participation is obtained, and a co-expression network of the experimental group and the control group is constructed by comparing the difference mT obtained in the experimental group and the control group, respectively constructing the co-expression network of the experimental group and the control group, potential core regulatory genes in both sets of samples were positioned by comparing differences on co-expression networks. 4. The expression of partial potential core regulatory genes, TLN1 and CCND2 in the endometrium of adenomyosis was detected by real-time fluorescence quantitative PCR and immunohistochemistry. Study results 1. There were 165 differences in the expression of CD44v6 and 61 2 differences in the expression of mCD44v6 in the endometrium of adenomyosis compared with the control endometrium. 117 of the differentially expressed CDcRNAs were down-regulated and 48 of them were upregulated. Among them, the most significant cRNA was n333955 (difference multiple: 0. 0032), and the most significant cRNA was n342839 (the difference was 4. 13). Of the differentially expressed mRNAs, the down-regulated nnn, the total number is 414, the total number of mbs up-regulated 198. Among them, the most significant mRNA was HBA1 (difference fold: 0. 02), and the most significant mRNA was THBS2 (the difference is 5. 14). The results showed that the expression level of n333955, n337373, n338909 was decreased in the endometrial tissue of adenomyosis, while n341651, n342794, n387706 expression levels were elevated, and the difference was statistically significant. The results of biological information analysis showed that there were 352 significant sexual functions including extracellular matrix composition, endothelial cell differentiation, intracellular signal transduction, positive regulation of cell migration, angiogenesis and so on. The remarkable sexual function of down-regulation differential gene participation was 283, including: gene expression, DNA replication, cell division, cell cycle G1/ S transition, immune response and so on. include MAPK signaling pathways, local adhesion, adhesion connections, extracellular matrix receptor interactions, extracellular matrix-Akt signaling pathways, and the like. There are 39 significant signal transduction pathways for down-regulation of differential gene participation, including DNA replication, cell cycle, primary immunodeficiency, cytokines, cytokine receptor interactions, and the like. 3. 3 constructs co-expression networks of two groups of samples, respectively, There are significant differences in the structure of the two co-expression networks. According to the change of the expression status of the genes in the two networks, the key mdr/ cGVHD, including TLN1, n342794, MYBL1, CCND2, ENST00000406939, n338909, etc., plays an important role in the sample difference. Real-time fluorescence quantitative PCR and immunohistochemistry showed that the expression of TLN1 gene and protein in endometrial tissue was significantly higher than that of control endometrial tissue, and the expression of CCND2 gene and protein in endometrial tissue was significantly lower than that of control endometrial tissue. The difference was statistically significant. Study conclusion 1. In this paper, the differential expression profiles of the endometrium of the adenomyosis and the control endometrium were successfully constructed by the chip detection. the real-time fluorescence quantitative PCR result has good consistency with the chip result, and further verifies the reliability of the chip result. By means of bioinformatic analysis, it is possible to preliminarily explore the possible regulative effect of mpr and mckcpr in the course of the occurrence of adenomyosis. The potential core regulatory genes TLN1 and CCND2 genes and proteins express abnormalities in the endometrial tissue of the adenomyosis.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R711.71

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