【作者】 张志平；

【导师】 吴建新；

【作者基本信息】 中国协和医科大学 ， 儿科学， 2010， 博士

【摘要】 神经管畸形(neural tube defects,NTDs)是由于在胚胎发育过程中,神经管闭合不全所引起的一类先天畸形。NTDs属多因素疾病,该病的发生主要是遗传因素和环境因素及两者相互作用的结果。这种畸形起因于神经管闭合的失败,所以影响神经管发育的因素都可能促使其形成。MicroRNAs (miRNAs)是一类长约22个核苷酸的内源性单链非编码RNA,通过与靶mRNA完全或不完全互补配对,导致靶mRNA降解或抑制其翻译,从而在基因的表达调控中发挥着重要作用。目前研究表明miRNAs在神经干细胞分化和哺乳动物脑发育过程中起重要作用,提示miRNAs可能参与神经管畸形的发生。但以往研究检测的miRNA数量有限,并且国内外尚无miRNA在胚胎脑组织中功能研究的报告。因此有必要深入开展miRNA在NTDs胚胎脑组织中的表达和功能研究,为深入了解NTDs的发病机理提供新的线索。本课题通过对于miRNAs在NTDs与正常胚胎脑组织的差异表达分析和特定miRNAs作用机制的阐释,有助于我们进一步提高对于NTDs发生发展的分子机制的认识。目的：建立神经管畸形与正常胚胎脑组织miRNAs的差异表达谱,利用生物信息学手段,预测差异表达显著的miRNAs的靶基因,并进一步分析靶基因的生物学功能及在神经管畸形发生中的作用。为进一步探讨miRNAs在神经管畸形发生机制中的作用提供依据。方法：利用1niRCURYTM LNA miRNA表达谱芯片分析无脑畸形残余脑组织和正常胎儿脑组织miRNAs差异表达。利用NCodeTM qRT-PCR kit试剂盒,以U6 snRNA作为内标,对差异表达显著的miRNAs进行验证。利用mirGEN v3数据库预测差异表达显著的9个miRNAs的靶基因,利用Human Protein Reference Database (HPRD)分析靶蛋白的相互作用,建立相互作用网。利用GeneCodis 2.0分析相互作用网所含蛋白质的生物学功能,并以此推断miRNAs在NTDs发生发展中的作用。结果：1. miRNA芯片检测发现：实验组与对照相比,有213个miRNAs表达差异显著(两组差异>2倍)。niRNAs表达上调2倍以上116个(其中包含1niRPLUS 9个),miRNAs表达下调2倍以上97个(其中包含miRPLUS 2个)2.依据表达差异显著性和在脑组织中表达的特异性,选择9个miRNAs进行real-time RT-PCR验证。miR-9、miR-212、miR-451、miR-198、miR-126、miR-124和miR-138检测结果与miRNA芯片一致,miR-103/miR-107与miRNA芯片结果相反。表达上调：miR-451、miR-198和miR-126,表达下调：miR-9、miR-212、miR-103、miR-107、miR-124和miR-138。这9个miRNAs在NTDs和正常对照中的表达差异均具有统计学意义(p<0.05)3.经过mirGEN v3数据库分析,881个基因被预测为这9个1niRNAs的靶基因。其中79个基因包含在蛋白质相互作用网中。4.经GeneCodis 2.0分析,这些蛋白参与基因转录、信号转导、细胞周期、细胞生长、死亡和细胞间信号转导这些重要的生物学过程；发挥蛋白结合、转录因子活化、受体结合及活化、核酸结合、DNA结合和转移酶活化等生物学功能；并且它们涉及肌动蛋白、Jak-STAT和MAPK等与神经发育相关的信号通路。结论：miRNAs在无脑畸形和正常对照胎儿脑组织中表达谱存在差异,可能参与了无脑畸形发生的分子机制。同时,本研究绘制了靶基因的蛋白质相互作用网,并分析了这些靶蛋白的生物学功能,为深入研究这些miRNAs的调控机制及在NTDs发生发展中的作用提供了基础。更多还原

【Abstract】 Neural tube defects (NTDs) are a group of severe congenital malformations characterized by a failure of neural tube closure during early embryonic development. NTDs are complex birth defects with a multi-factorial pattern of inheritance, requiring both genetic and environmental factors to contribute to their etiology.NTDs are caused by the failure of the neural tube to close. Therefore, factors that impact the development of the neural tube are likely to be involved in the pathogenesis of NTDs. MicroRNAs (miRNAs) are small, single-stranded, and non-coding RNA molecules that possess about 22 nucleotides. The function of miRNAs in mammalian cells is to repress the translation or cleavage of target gene messenger RNA (mRNA) by base-pairing with the 3’-untranslated regions (UTRs) of target mRNAs.A number of miRNA profiling studies have shown that the expression of miRNAs changes during neural stem cell differentiation and during the morphological development of the mammalian brain. These researches suggest that miRNAs may be important players in these processes. However, neither the expression nor the role of miRNAs in NTDs has been characterized. Thus, it is necessary to unravel the expression and function of the miRNAs in NTDs. Our study may provide a further understand the etiology of NTDs.Objective:To investigate the expression of miRNAs in tissues from fetuses with the most severe NTDs, anencephaly and normal control, and discuss the roles of miRNAs in the pathogenesis of NTDs. To predict the target genes of miRNAs which the expression change was confirmed and analysis the function of target genes.Methods:The profiling of miRNAs from area cerebrovasculosa of fetuses with anencephaly and normal brains was performed using an LNA mercuryTM microarray. Real-time RT-PCR analysis was carried out using an NCodeTM miRNA First-Strand cDNA Synthesis and a qRT-PCR kit. Relative expression was calculated using the AACT method and normalizing to the expression of U6 snRNA.The target gene sets for miRNAs were determined using miRGen v3. The human protein interaction data set was obtained from the Human Protein Reference Database (HPRD).Using these data, we constructed a protein interaction network of the predicted miRNA target genes. GeneCodis 2.0 was used to search for biological features of the interacting proteins involved in the network.Results:1. Compared to healthy human fetal brain tissue, tissue from fetuses with anencephaly has a specific miRNA expression profile. In anencephaly, there were 97 downregulated miRNAs and 116 upregulated miRNAs. These miRNAs include two downregulated miRPlus and nine upregulated miRPlus.2. The microarray findings were extended using real-time qRT-PCR for nine miRNAs. Of these nine miRNAs validated, miR-126, miR-198, and miR-451 were upregulated, whereas miR-9, miR-212, miR-124, miR-138, and miR-103/107 were downregulated in the tissues from fetuses with anencephaly.3. Bioinformatic analysis shows 881 potential target genes that are regulated by the validated miRNAs. Of these potential genes,79 genes are involved in a protein interaction network.4. There are six co-occurrence annotations (transcription, signal transduction, cell cycle, cell growth, cell death, and cell-cell signaling) within the GOSlim Process. There are seven co-occurrence annotations (protein binding, transcription factor activity, receptor activity, Nucleotide binding, DNA binding, receptor binding, and transferase activity) within the GOSlim Function found by Genecodis2. And there are thirty-five co-occurrence annotations (such as focal adhesion,actin cytoskeleton and Jak-STAT signaling pathway et al.) within the KEGG pathway found by Genecodis2.Conclusion:This study confirms that anencephaly has a specific miRNA expression profile compared to gestational age-matched normal fetal brain tissue. The results show that different miRNAs are deregulated in NTDs, suggesting the involvement of these miRNAs in the pathogenesis of NTDs. We also provide a target gene map of miRNAs that are predicted to contribute to future investigations of the regulatory mechanisms of miRNA in human anencephaly.更多还原