形觉剥夺性近视相关miRNA的生物信息学分析及功能预测

发布时间：2018-01-10 03:11

本文关键词：形觉剥夺性近视相关miRNA的生物信息学分析及功能预测　出处：《南京医科大学》2017年博士论文　论文类型：学位论文

【摘要】：近视是影响全球的健康问题,根据资料推算,到2050年,全世界将有接近一半人口近视,并且全世界人口的1/10将会高度近视(高于600度近视)。在过去的几十年中,近视发病率迅速增加,在很多东亚和东南亚国家的高中毕业生中近视发生率高达80%～90%,高度近视发生率达10～20%,已经成为一个重大的公共健康问题。在世界其他地方,比如北美、欧洲和中东,尽管情况不如亚洲严重,但近视发病率也在逐年上升。虽然可以通过配戴眼镜、隐形眼镜或者进行屈光手术的方法矫正屈光不正,但由于筛查手段不健全、治疗手段不实用或者缺乏经济能力等原因,近视仍然是目前视力损害的主要原因。此外,逐渐加深的近视还会增加视网膜脱离、白内障、青光眼,甚至失明的风险。对近视的有效预防和治疗应该建立在充分了解其发病机制的基础之上。然而,尽管全球众多学者多年来做了大量的研究,但是其具体发病机制至今仍不能明确。一般认为在形觉剥夺性近视(Form deprivation myopia,FDM)模型中主要通过两种机制起作用,即"巩膜主动重塑机制"和"局部视网膜调控机制",两者均认为外界刺激作用于视网膜,启动视网膜-视网膜色素上皮层-脉络膜信号转导系统,把局部视网膜刺激信号转化为调控巩膜主动重塑的信号,诱导细胞外基质表达异常,产生巩膜胶原纤维改变等变化,引起巩膜主动重塑,最终导致眼轴长度与屈光状态不匹配,形成近视,引起视功能减退及各种并发症。研究发现,在巩膜主动重塑过程中,miRNA起到重要信号传导和调控作用。miRNA是一种小的非编码单链RNA,在转录后水平上起着调控基因表达的重要作用。这种调控是通过与目标基因3'非翻译区(UTR)碱基配对的形式实现的,通过完美或近乎完美的配对(植物)或者不全配对(哺乳动物)产生翻译抑制,导致mRNA裂解和降解。因此,miRNA可以作为信号网络中的节点,通过调控基因表达量的变化,实现调节细胞的增殖、分化、代谢和凋亡等多种细胞活性的功能。目前已有大量研究报道miRNA参与人体正常和疾病状态过程,并在其中起到重要作用。研究发现眼部组织,如视网膜、晶状体、角膜组织中均有miRNA的转录。只有极少数miRNA功能已知,而绝大多数miRNA在正常组织和疾病过程中的作用仍不明确。目前公认miRNA在非眼部和眼组织的正常生理和病理过程中起着至关重要的作用,以往研究发现在正常眼生长过程中(眼轴伸长),年轻且快速增长的眼球与那些成年稳定(假定)的眼球巩膜组织中的相关性miRNAs表达差异显著。一些差异表达的miRNAs是活跃的巩膜外细胞重塑和变薄的产物,可能与细胞外基质重塑途径有关,从而使其成为预防或延缓近视进展的潜在靶点。作为探索潜在miRNA作为控制近视治疗靶点的第一步,本研究试图了解他们在近视发生过程中对巩膜变化的作用,因为我们认为眼轴伸长(生长)形成近视的发展过程直接与巩膜miRNA的差异调节有关。本研究以从已知的基因表达资料库下载完整的配对良好的形觉剥夺性近视(FDM)小鼠模型miRNA芯片检测数据为研究对象,采用生物信息学方法,探索与形觉剥夺性近视(FDM)高度相关的miRNA;再通过miRDB在线工具寻找与这些miRNA密切相关的基因;最后通过DAVID数据库预测这此miRNA可能的功能。本研究旨在通过生物信息学方法寻找调控小鼠形觉剥夺性近视(FDM)发生发展过程的重要miRNA,预测其功能,为进一步实验研究指明目标和方向。本研究将从如下三部分分析形觉剥夺性近视(FDM)相关miRNA的生物学功能:第一部分形觉剥夺性近视相关miRNA生物信息学分析目的:探索和寻找与形觉剥夺性近视(FDM)形成过程密切相关的miRNA。方法:在公共基因芯片数据库(Gene Expression Omnibus,GEO)中检索并选取设计合理、资料完整的形觉剥夺性近视(FDM)小鼠模型相关miRNA芯片表达谱数据(GSE58124)。数据检测组织为小鼠全眼组织、巩膜组织和视网膜组织,同一小鼠右眼为实验组,左眼为对照组,共纳入样本24个,6个全眼组织对照样本、6个形觉剥夺性近视(FDM)全眼组织样本;3个巩膜组织对照样本、3个形觉剥夺性近视(FDM)巩膜组织样本;3个视网膜组织对照样本、3个形觉剥夺性近视(FDM)视网膜组织样本。经过数据预处理,使用中值法进行片间标准化,获得单个数据矩阵的标准化后的矩阵。分别对三组配对的实验组和对照组数据进行分析,取| logFC(FC,foldchange)|1且p0.05为入选标准,筛选得到在形觉剥夺性近视(FDM)模型视网膜组织、全眼组织和巩膜组织中有显著差异的miRNA(上调或者下调),从中得到与形觉剥夺性近视(FDM)形成过程高度相关的miRNA。结果:经过数据处理和分析,最终筛选得到视网膜组织中24个显著差异miRNA(均为上调)、全眼组织中20个显著差异miRNA(均为上调)、巩膜组织中没有符合条件的miRNA,经过比对发现有8个miRNA在视网膜组织和全眼组织中均显著上调。结论:至少有 8 个 miRNA(mmu-miR-468,mmu-miR-16-1*,mmu-miR-466h-5p,mmu-miR-466j,mmu-miR-669e,mmu-miR-15a*,mmu-miR-466c-5p-v15.0和mmu-miR-294)参与调节小鼠形觉剥夺性近视(FDM)形成过程。第二部分形觉剥夺性近视相关miRNA靶基因预测目的:预测8个共同差异表达的miRNA的下游调控靶基因。方法:运用在线数据库软件miRDB进行8个共同差异表达miRNA的下游调控靶基因预测。结果:经过miRDB在线数据库检索,8个共同差异表达miRNA总共预测到1805个下游靶基因,其中2个靶基因被5个miRNA共同调控,5个靶基因被4个miRNA共同调控,82个靶基因被3个miRNA共同调控;共89个miRNA被至少三个miRNA共同调控。结论:7个至少被4个共同差异表达miRNA共同调控的靶基因分别为REEP3、MAPK10、INO80D、D630045J12RIK、FMR1、ARMC8 和 BACH2,它们可能是参与调控形觉剥夺性近视(FDM)的主要目的基因。第三部分形觉剥夺性近视相关miRNA功能预测目的:预测8个共同差异表达的miRNA的功能。方法:运用在线数据库软件 DAVID(The Database for Annotation,Visualization and Integrated Discovery)对8个共同差异表达miRNA预测的靶基因进行功能富集分析来研究差异表达miRNA的功能。结果:8个共同差异表达miRNA的靶基因通路富集分析结果发现在细胞通路层面上有5个miRNA的靶基因显著的富集到了 mmu04360:Axon guidance通路(mmu-miR-16-1*、mmu-miR-294、mmu-miR-466c-5p、mmu-miR-466j 和 mmu-miR-468),4个miRNA 的靶基因显著的富集到了 mmu04350:TGF-betasignaling pathway(mmu-miR-16-1*、mmu-miR-466j、mmu-miR-669e 和 mmu-miR-294)。在基因本体论层面上,GO:0045449～regulation of transcription被7个差异表达的miRNA 的靶基因所富集到,GO:0006350～transcription/GO:0006355～regulation of transcription,DNA-dependent/GO:0051252～regulation of RNA metabolic process 这4个GO BP条目被6个差异表达的miRNA的靶基因所共同富集到。mmu-miR-466h-5p与mmu-miR-466j的靶基因有非常多的共同富集到的GOBP条目,说明两个 miRNA 之间的功能协作性。mmu-miR-294、mmu-miR-16-1*、mmu-miR-669e和mmu-miR-466c-5p这4个miRNA也有较多的功能协作。结论:8个共同差异表达的miRNA通过对靶基因的调控,干扰多个细胞通路或生物过程,从而在近视发展过程中发挥作用。结论1.miRNA或许并没有在形觉剥夺性近视(FDM)小鼠的巩膜组织中起到重要影响作用。2.在对全眼组织和视网膜组织的研究中发现8个显著的共同表达上调的miRNA(mmu-miR-468,mmu-miR-16-1*,mmu-miR-466h-5p,mmu-miR-466j,mmu-miR-669e,mmu-miR-15a*,mmu-miR-466c-5p-v15.0 和 mmu-miR-294).3.miRNA 通过调控 REEP3、MAPK10、INO80D、D630045J12RIK、FMR1、ARMC8和BACH2等靶基因参与形觉剥夺性近视(FDM)的发展过程。4.通过对miRNA靶基因功能的预测分析显示"转录调控"显著增强,丰富了"轴突导向"和"TGF-β信号通路"被广泛涉及,mir-466h-5p和mir-466j显著富集在突触传递相关的生物过程中。
[Abstract]:Myopia is a global health problem, according to the projections, by 2050, the world will have nearly half of the population of the world's population myopia, and high myopia (1/10 will be higher than 600 degrees of myopia). In the past few decades, the incidence rate of myopia increased rapidly in many East Asian and Southeast Asian countries in the high school graduates the incidence of myopia is as high as 80% to 90%, high myopia rate ranging from 10 to 20%, has become a major public health problem in the world. In other places, such as North America, Europe and the Middle East, although not in Asia but serious, the incidence of myopia is increasing year by year. Although you can wear glasses, contact lenses and methods or refractive surgery for correction of ametropia, but because screening is not perfect, treatment is not practical or lack of economic capacity and other reasons, myopia remains the leading cause of visual impairment. In addition, The deepening of myopia will increase retinal detachment, cataract, glaucoma, risk and even blindness. On the basis of effective prevention and treatment of myopia should be based on the full understanding of its pathogenesis. However, although many of the world's many years scholars have done a lot of research, but the mechanism is still not clear. In general form deprivation myopia (Form deprivation, myopia, FDM) mainly through two kinds of mechanisms in the model, namely "scleral remodeling mechanism" and "local retinal mechanism", both think outside stimulus effects on the retina, start the retina retinal pigment epithelium choroid to retina local signal transduction system. The stimulus signal into signal regulation in scleral remodeling, abnormal expression induced by extracellular matrix, produce scleral collagenous fibres change caused by changes in scleral remodeling The final result, axial length and refractive status does not match, the formation of myopia, visual hypofunction and various complications. The study found that in the process of scleral remodeling, miRNA plays an important role in signal transduction and regulation of.MiRNA is a small non encoding single stranded RNA, plays an important role in the regulation of gene expression in post transcriptional level. This regulation is through the target gene and 3'untranslated region (UTR) to realize the base pairing form, through perfect or near perfect pairing (plant) or incomplete pairing (mammalian) generated translation inhibition, leading to mRNA cleavage and degradation. Therefore, miRNA can be used as a signal node in the network, expression through the change of gene regulation, regulate cell proliferation, differentiation, metabolism and apoptosis of various cell activity functions. Many studies have reported miRNA in normal and disease of human body and in the process. Play an important role. The study found that the eye tissues, such as retina, lens, transcription of miRNA were in the corneal tissue. Only a handful of miRNA functions are known, and most of the miRNA function in normal tissues and disease are still not clear. The normal physiological and pathological processes as miRNA in non eye and eye tissue. Which plays a vital role in previous studies found in the normal eye growth process (axial elongation), young and the rapid growth of the eyeball and those of adult stable (assuming) miRNAs correlation of sclera tissue expression significant difference. Some differences between the expression of miRNAs is reshaping the scleral active cells and thinning of the product that may be associated with extracellular matrix remodeling pathway, which makes it a potential target to prevent or delay the progression of myopia. As the exploration potential miRNA as a first step to control myopia therapeutic targets, This study attempts to understand them in the process of the occurrence of myopia scleral changes, because we believe that the axial elongation (growth) during the formation and development of myopia and direct scleral miRNA differentially regulated. In this study, from the known gene expression database to download the complete good pairing of form deprivation myopia (FDM) mouse model miRNA microarray data as the research object, using bioinformatics methods, explore and form deprivation myopia (FDM) was highly correlated with miRNA; and then through the miRDB online tools to find those closely related to miRNA gene; finally through the DAVID database to predict the function of miRNA. This may be the purpose of this study is to search through bioinformatics methods control mice form deprivation myopia (FDM) is an important development of miRNA process, predict its function for further study indicates the target and direction. This research will be from the following The three part is the analysis of form deprivation myopia (FDM) related to the biological function of miRNA: in the first part of form deprivation myopia related bioinformatic analysis of miRNA Objective: To explore and find and form deprivation myopia (FDM) to form miRNA. process is closely related to the public: in the gene chip database (Gene Expression Omnibus, GEO) in the search and select the reasonable design, complete data form deprivation myopia (FDM) expression data related to mouse model of miRNA chip (GSE58124). Data organization for the detection mouse eye tissue, sclera and retina tissue, with a right mouse as the experimental group, the left eye as control group, were included in the sample 24, 6 full eye tissue control samples, 6 form deprivation myopia (FDM) ocular tissue samples; 3 scleral tissue control samples, 3 form deprivation myopia (FDM) the 3 kind of scleral tissue; retinal tissue control samples, 3 Form deprivation myopia (FDM) retinal tissue samples. After data preprocessing, chip standardization using median method, matrix single data matrix after standardization. Experimental group of three groups and paired control group data were analyzed from | logFC (FC, foldchange) |1 and P0.05 the inclusion criteria, screened in form deprivation myopia (FDM) retinal tissue model, there are significant differences of eye tissue and scleral tissue in miRNA (increasing or decreasing), and form deprivation myopia (FDM) formed from miRNA. results highly correlated process: after data processing and analysis, the final selection 24 significant differences in retinal tissue (miRNA were up-regulated), 20 significant differences miRNA ocular tissues (all up-regulated), scleral tissue did not meet the conditions for miRNA, after comparison found that 8 miRNA in the retina and ocular tissue Were upregulated. Conclusion: there are at least 8 miRNA (mmu-miR-468, mmu-miR-16-1*, mmu-miR-466h-5p, mmu-miR-466j, mmu-miR-669e, mmu-miR-15a*, mmu-miR-466c-5p-v15.0 and mmu-miR-294) were involved in the regulation of form deprivation myopia (FDM) formation process. The second part form deprivation myopia related miRNA target gene prediction to downstream target gene prediction expression 8 a common difference of miRNA. Methods: using online database software miRDB expression predicts downstream target gene miRNA 8 common difference. Results: after miRDB online database retrieval, miRNA a total of 1805 predicted target gene expression differences of 8 common, 2 of which target gene is 5 miRNA common control the 5 target gene is 4 miRNA regulated, 82 genes were 3 miRNA common control; a total of 89 miRNA was at least three miRNA common control. Conclusion: 7 by at least 4 The target gene miRNA to regulate expression of common differences were REEP3, MAPK10, INO80D, D630045J12RIK, FMR1, ARMC8 and BACH2, they may be involved in the regulation of form deprivation myopia (FDM) gene. The third part is the main purpose of form deprivation myopia related miRNA function prediction Objective: to predict the expression of 8 common difference the function of miRNA. Methods: using online database software DAVID (The Database for Annotation Visualization and Integrated Discovery) expression of functional enrichment analysis to study the differential expression of miRNA function of the miRNA target gene prediction of 8 common differences. Results: the target miRNA gene pathway enrichment analysis showed that the target gene has 5 miRNA in cells the level of significant enrichment pathway to mmu04360:Axon guidance pathway 8 common difference (mmu-miR-16-1*, mmu-miR-294, mmu-miR-466c-5p, mmu-miR-466j and mmu- MiR-468), the target gene 4 miRNA significant enrichment to mmu04350:TGF-betasignaling pathway (mmu-miR-16-1*, mmu-miR-466j, mmu-miR-669e and mmu-miR-294). The gene ontology level, GO:0045449 ~ regulation of transcription is the target gene 7 differentially expressed by miRNA enrichment, GO:0006350 ~ transcription/GO:0006355 ~ regulation ~ of transcription, DNA-dependent/GO:0051252 regulation of RNA metabolic process 4 GO BP entries were target genes of 6 differentially expressed miRNA common enrichment to target genes.Mmu-miR-466h-5p and mmu-miR-466j have a lot of common enrichment to GOBP entries, that function collaboration between two miRNA.Mmu-miR-294, mmu-miR-16-1*, mmu-miR-669e and mmu-miR-466c-5p 4 miRNA also has the function of collaboration more. Conclusion: the expression of 8 common differential miRNA through the target Gene regulation, interference of multiple cell signaling or biological process, which play a role in the development of myopia in the process. Conclusion 1.miRNA may not in form deprivation myopia (FDM) plays an important role in.2. found 8 significant co expression upregulation of miRNA in the study of the whole eye tissue and retinal tissue to tissue sclera in mice (mmu-miR-468, mmu-miR-16-1*, mmu-miR-466h-5p, mmu-miR-466j, mmu-miR-669e, mmu-miR-15a*, mmu-miR-466c-5p-v15.0 and mmu-miR-294) of.3.miRNA MAPK10, through regulating REEP3, INO80D, D630045J12RIK, FMR1, ARMC8 and BACH2 target genes involved in form deprivation myopia (FDM) in the development process of.4. through the prediction of miRNA target gene function analysis showed that "transcriptional regulation" was significantly enhanced and enriched "axon guidance" and "TGF- beta signaling pathway is widely involved, mir-466h-5p and mir-466j were significantly enriched in synaptic transmission. In the biological process of closing.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R778.11

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