人ESC分化成RPE过程中的microRNA序列和表达模式的多样性的研究

发布时间：2018-04-08 15:44

本文选题：人　切入点：胚胎干细胞　出处：《湖南科技大学》2015年硕士论文

【摘要】：年龄性黄斑变性(AMD)是全世界范围内老年人视力受损和丧失的首要原因。AMD是一种视网膜黄斑变性的眼疾,至今都没有特效治疗手段。近年来发展了用干细胞衍生视网膜色素上皮细胞(RPE)作为AMD替代性治疗的新方案。胚胎干细胞(ESC)分化成RPE的过程中涉及很多基因的表达变化,包括micro RNA(mi RNA)的表达变化。为了揭示mi RNA在这个细胞分化转变过程的变化和作用,本研究首次系统地研究了人类ESC分化成RPE的四个阶段的mi RNA的表达水平变化、阶段特异性表达的mi RNA、不同分化阶段优势表达臂转换的mi RNA与isomi R中编辑位点位于“seed”区的mi RNA的潜在功能变化,即在细胞分化中的mi RNA的表达水平、表达模式和序列多样性,以及相关功能等四个方面的变化。研究结果具体体现在以下方面:1.在干细胞分化为RPE细胞的四个阶段中,检测到了941个已知的mi RNA前体,发现了113个新的mi RNA发卡及其表达的196个mi RNA。2.与干细胞分化的第一阶段相比较,在第四阶段显著上调的mi RNA有205个,下调的有295个,第二阶段有172个显著上调和175个显著下调的mi RNA,在第三阶段有227个显著上调和331个显著下调的mi RNA。另外,三个ESC已分化的阶段中表达模式一致的有93个显著上调和124个显著下调的mi RNA。分析第四阶段已分化的RPE细胞与ESC细胞间的差异表达mi RNA大致的相关功能后,发现大多数在RPE细胞中下调mi RNA的靶标的最富集的GO术语和上调mi RNA靶标的相类似,因为它们享有各自大部分的靶标集。3.发现了107个优势表达的、与mi RBase中记录的标准的mi RNA序列不一致的5′-isomi R序列,而在所有阶段所有细胞都优势表达的isomi R共有19个;这19个优势表达的5′-isomi R与其对应的标准型mi RNA的功能分析结果表明尽管它们之间分享超过一半的过度呈现的生物学过程(P-value0.01),但这两组mi RNA还是各自参与了明显不同生物过程。4.在24个不同的mi RNA中鉴定了27个统计显著性的A-to-I编辑位点,这些位点均不是已知的SNP位点;绝大多数这些位点位于成熟mi RNA的“seed”区,并且除了hsa-mi R-381-3p中的编辑事件发生在ESC细胞分化的早期,其它的均发生在ESC细胞分化的晚期。有九个推断的A-to-I编辑位点得到了已有实验结果的验证。同时发现编辑mi RNA和相应的未编辑mi RNA的功能存在很大的变化。5.人类ESC分化成RPE的各阶段间有14个mi RNA前体产生的mi RNA存在明显的臂选择倾向性表达的这种现象。这14个臂转换的pre-mi RNA共表达了27个mi RNA。mi RNA靶标的功能分析表明在ESC分化为RPE细胞过程中的这些优势表达臂转换mi RNA与ESC的维持或ESC的分化相关。
[Abstract]:Age-related macular degeneration (AMDD) is the leading cause of visual impairment and loss in the elderly worldwide. AMD is an eye disease with macular degeneration.In recent years, stem-derived retinal pigment epithelial cells (RPE) have been developed as a new alternative therapy for AMD.The differentiation of embryonic stem cells into RPE involves changes in the expression of many genes, including the expression of micro RNA(mi.In order to reveal the changes and roles of mi RNA in the process of cell differentiation and transformation, this study has for the first time systematically studied the changes in the expression level of mi RNA in the four stages of differentiation of human ESC into RPE.In different differentiation stages, the potential functional changes of mi RNA and isomi R where the editing site is located in "seed" region, that is, the expression level of mi RNA in cell differentiation, are expressed at different differentiation stages.The diversity of expression patterns and sequences, as well as the changes of related functions.The results are reflected in the following aspects: 1. 1.In the four stages of stem cell differentiation into RPE cells, 941 known RNA precursors were detected, 113 new mi RNA hairpins and 196 miRNA.2expressed.Compared with the first stage of stem cell differentiation, 205 mi RNA were significantly up-regulated and 295 down-regulated in the fourth stage.In the second stage, there were 172 significantly up-regulated and 175 down-regulated mi RNAs, while in the third stage there were 227 significantly up-regulated and 331 significantly down-regulated mi RNAs.In addition, 93 up-regulated and 124 down-regulated miRNAs were found in the three differentiated stages of ESC.After analyzing the differential expression of mi RNA between differentiated RPE cells and ESC cells in the fourth stage, it was found that most of the most enriched go terms down-regulating the target of mi RNA in RPE cells were similar to those that up-regulated the target of mi RNA.Because they have most of their target set. 3.107 preferentially expressed 5'-isomi R sequences inconsistent with the standard mi RNA sequences recorded in mi RBase were found, while 19 isomi R preferentially expressed in all cells at all stages;The functional analysis of the 19 preferentially expressed 5'-isomi R and its corresponding standard type mi RNA showed that although they shared more than half of the overrepresented biological processes (P-value0.01), the two groups of mi RNA were involved in different biological processes respectively. 4.Twenty-seven statistically significant A-to-I editing sites were identified in 24 different mi RNA, none of which are known SNP sites. The majority of these sites are located in the "seed" region of the mature mi RNA.In addition, the editing events in hsa-mi R-381-3p occurred in the early stage of ESC cell differentiation, and others occurred in the late stage of ESC cell differentiation.Nine inferred A-to-I editing sites have been verified by existing experimental results.At the same time, it is found that the functions of edit mi RNA and corresponding unedited mi RNA have great changes.During the process of human ESC differentiation into RPE, there are 14 mi RNA precursors producing mi RNA, which has the phenomenon of brachial selective tendency expression.The functional analysis of 27 mi RNA.mi RNA targets expressed by the 14 arm converted pre-mi RNA showed that these advantages in the process of ESC differentiation into RPE cells were related to the maintenance of ESC or the differentiation of ESC.
【学位授予单位】：湖南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R774.5

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