卵巢子宫内膜异位症中1ncRNA表达谱及HOXA9，HOXA10，HOXA11和HOXD10的甲基化水平研究

发布时间：2018-02-28 23:13

本文关键词： 子宫内膜异位症 1ncRNA mRNA 子宫内膜异位症生物信息学分析 1ncRNA qRT-PCR HOXA9 HOXA10 HOXA11 HOXD10 DNA甲基化　出处：《北京协和医学院》2014年博士论文　论文类型：学位论文

【摘要】：虽然目前子宫内膜异位症的病因仍然不明,但一般认为它是一种多因素共同导致的疾病,而其中遗传因素,尤其是表观遗传学调控在其中起到了重要的作用。比如,DNA甲基化水平改变参与了在内异症中有重要作用的HOXA10和HOXA11基因在在位内膜中的表达下调。HOXA9和HOXD10基因则被发现与HOXA10和HOXA11在功能和表达模式上高度一致。而在本课题组的前期研究中,我们发现外周血microRNA有作为内异症生物学标记物的潜能。长链非编码RNA(long non-coding RNA,lncRNA)作为转录组的重要组成部分,在人类疾病、发育、干细胞等领域发挥了重要的调控作用。而对1ncRNA在内异症中表达情况的研究尚属空白。因此,在本课题中,我们利用微阵列基因芯片技术,探索1ncRNA在卵巢内异症组织和在位内膜组织中的表达差异,建立1ncRNA的表达谱。在此基础上,使用生物信息学方法预测1ncRNA的潜在功能。另外,在本研究中我们还探索了DNA甲基化水平异常是否参与了HOXA10,HOXA11, HOXA9和HOXD10基因在内异症组织中相对于在位内膜的表达下调。研究目的：在全基因组范围内比较卵巢子宫内膜异位症患者在位及异位内膜组织中lncRNA的表达水平,建立lncRNA的表达谱。研究方法：使用高通量的微阵列基因芯片,对4对卵巢内异症患者的在位及异位内膜组织同时检测全基因组范围内lncRNA和mRNA的表达水平,筛查在位及异位内膜组织中差异表达的lncRNA和mRNA,获得内异症中lncRNA的表达谱。研究结果：相对于在位内膜,卵巢内异症组织有4,088个mRNA转录本和948个lncRNA转录本出现了差异表达。研究结论：相对于在位内膜组织,mRNA和lncRNA在卵巢内异症组织中表现出了系统性表达差异。实验二部分差异表达的lncRNA的功能预测及PCR验证研究目的：进一步探索1ncRNA在子宫内膜异位症中可能发挥的功能,同时验证实验一基因芯片结果的可信性。研究方法：利用已有的mRNA的功能注释,预测差异表达的lncRNA的功能。在实验一基础上,扩大检测样本量,在21对卵巢内异症患者的在位及异位内膜组织中,对2组共10个实验一基因芯片结果中差异表达的lncRNA进行实时定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测,以对基因芯片结果进行验证。研究结果：差异表达的1ncRNA可能参与到了多种生物学通路中,其中包括数种已知的内异症发病机制中的重要通路。1ncRNA可能通过顺式和反式两种作用方式调控编码蛋白的基因,从而发挥作用。经qRT-PCR验证,lncRNA FN0206、MGC24103、 LOC375295、CHL1-AS2、XL0C_012981、LOC375295和CHL1-AS2表达上调;LIMS3-LOC440895、LOC389906、HOXA11-AS、KLKP1、LOC100505776和XLOC_012981表达下调。研究结论：1ncRNA可能通过多种生物学通路参与到内异症的发病机制中。实验一使用基因芯片检测1ncRNA表达的结果可靠。实验三部分卵巢内异症患者在位与异位内膜中HOXA9、HOXA10、HOXA11和HOXD10基因甲基化状态的研究研究目的：研究DNA甲基化水平的改变是否可能是导致HOXA9、HOXA10、HOXA11和HOXD10基因在内异症组织中表达失调的潜在原因。研究方法：对HOXA9、HOXA10、HOXA11和HOXD10基因,首先使用qRT-PCR技术在20对卵巢内异症在位及异位内膜样本中检测其表达水平。接下来,使用甲基化芯片技术,在5对样本中检测它们启动子区的甲基化水平。研究结果：HOXA9、HOXA10、HOXA11和HOXD10基因在异位内膜组织中的表达下调在qRT—PCR实验中得到了验证,HOXA10基因CpG岛1在异位内膜中出现了低甲基化,其他基因未发现启动子区甲基化水平的明显变化。研究结论：HOXA9、HOXA10、HOXA11和HOXD10基因启动子区的甲基化水平变化可能不是导致卵巢内异症组织与在位内膜组织间表达水平差异的主要原因。
[Abstract]:Although the cause of endometriosis remains unknown, but it is generally considered a disease caused by multiple factors, including genetic factors, especially the epigenetic regulation has important effect in them. For example, the level of DNA methylation changes in the disease, abnormal expression of HOXA10 gene and HOXA11 gene an important role in the endometrial down-regulation of.HOXA9 and HOXD10 genes were found with HOXA10 and HOXA11 in the expression patterns and function is highly consistent. In ourprevious study, we found that the peripheral blood microRNA of endometriosis as biological markers of potential.
Long chain encoding RNA (long non-coding non RNA, lncRNA) as an important part of the transcriptome development in human disease, and play an important role in the regulation of stem cells and other fields. And the study of 1ncRNA expression in endometriosis is still blank. Therefore, in this study, we used microarray gene chip technology, to explore the differences of expression of 1ncRNA in ovarian endometriosis tissue and eutopic endometrial tissues, expression of 1ncRNA spectrum. On this basis, the potential function of the use of bioinformatics prediction methodology of 1ncRNA.
In addition, in this study, we also explored whether the abnormal DNA methylation level was involved in the downregulation of HOXA10, HOXA11, HOXA9 and HOXD10 genes in endometriosis tissues compared with the eutopic endometrium.
Research purposes: To compare the expression level of lncRNA in eutopic and ectopic endometrium of patients with ovarian endometriosis in the whole genome, and to establish lncRNA expression profile.
Methods: using high throughput microarray gene chip, and to detect the expression of genome wide lncRNA and mRNA of 4 patients with ovarian endometriosis eutopic and ectopic endometrial tissue, differential expression screening of eutopic and ectopic endometrium tissues of lncRNA and mRNA, the expression of lncRNA in different gain spectrum of the disease.
Results: compared with the eutopic endometrium, 4088 mRNA transcripts and 948 lncRNA transcripts showed differential expression in the ovarian endometrium.
Conclusion: compared with the eutopic endometrium, mRNA and lncRNA showed systematic expression differences in the ovarian endometrium.
The functional prediction and PCR verification of the differential expression of lncRNA in the two part of the experiment
Objective: to further explore the possible function of 1ncRNA in endometriosis, and to verify the credibility of the results of a gene chip.
Research methods: using the functional annotation of existing mRNA, predicted the differentially expressed lncRNA functions. In the experimental basis, enlarge the sample detection, in 21 of patients with ovarian endometriosis eutopic and ectopic endometrial tissue, differential expression of 2 groups of a total of 10 experimental results of gene chip lncRNA real-time quantitative polymerase chain reaction (Quantitative real-time polymerase chain reaction, qRT-PCR) to detect, to validate the results of gene chip.
Results: expression of 1ncRNA may be involved in a variety of biological pathways, including.1ncRNA pathway of endometriosis pathogenesis of a number of known in May by the cis - and trans gene two protein encoding regulation mode of action, so as to play a role. Verified by qRT-PCR, lncRNA FN0206, MGC24103, LOC375295, CHL1-AS2, XL0C_012981, upregulation of LOC375295 and CHL1-AS2; LIMS3-LOC440895, LOC389906, HOXA11-AS, KLKP1, LOC100505776 and XLOC_012981 expression.
Conclusion: 1ncRNA may be involved in the pathogenesis of endometriosis through various biological pathways. In Experiment 1, the detection of 1ncRNA expression by gene chip is reliable.
Study on the methylation status of HOXA9, HOXA10, HOXA11 and HOXD10 genes in the three part of experimental ovarian endometriosis patients
Objective: To study whether the change of DNA methylation level may be the underlying cause of HOXA9, HOXA10, HOXA11 and HOXD10 gene expression imbalance in endometriosis.
Methods: HOXA9, HOXA10, HOXA11 and HOXD10 genes, the first use of qRT-PCR technology in 20 to detect the expression of ovarian endometriosis eutopic and ectopic endometrial samples. Then, the use of chip technology in the detection of methylation, 5 methylation level of promoter region of their sample.
Results: the down regulated expression of HOXA9, HOXA10, HOXA11 and HOXD10 genes in ectopic endometrium was verified in the qRT PCR experiment. HOXA10 gene CpG island 1 showed hypomethylation in ectopic endometrium, and no significant change in promoter methylation level was found in other genes.
Conclusion: the methylation level of promoter region of HOXA9, HOXA10, HOXA11 and HOXD10 may not be the main reason for the difference in expression level between ovarian endometriosis and eutopic endometrium.

【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】：2014
【分类号】：R711.71

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