逆转座子LINE-1调控靶基因的鉴定及机制研究

发布时间：2018-12-10 21:18

【摘要】：近年来研究发现真核生物基因组中有许多重复序列在基因的表达调控具有重要的作用。其中很大一部分是非编码序列,其对编码基因的转录、翻译和表达等过程中有着重要的调控作用。这些可动元件的活化可改变邻近基因的表达,在编码区内或旁边的新插入点能够引起突变并导致基因表达发生变化和基因组的重塑。在这些重复序列中,有一种广泛分布于基因组中的逆转座子LINE-1,目前发现其对细胞的增殖与分化以及肿瘤的发生起着非常重要的作用。LINE-1是人类基因组中的主导转座子,在正常分化的细胞中,LINE-1的启动子为高度甲基化状态,造成其沉默,但生殖细胞发育、胚胎发育的早期以及肿瘤细胞中则为激活状态。研究表明在几乎所有的肿瘤细胞中LINE-1均为激活状态,提示其激活可能涉及肿瘤发生的共同机制。所以通过LINE-1的研究对了解肿瘤发生、发展和调控的分子机理以及肿瘤药物靶标具有重要意义,同时也为开发新型的肿瘤治疗药物提供新的靶点 LINE-1全长基因包括两个开放阅读框ORF1和ORF2,表达两种蛋白ORFlp和ORF2p,相对于ORF2p, ORF1p的表达大大过量。其中ORFlp具有与自身核酸结合活性,形成核酸蛋白复合物(RNP),而ORF2p具有内核酸酶及外切酶活性,与ORF1p一起执行逆转座功能,其机制为TPRT (Target-primed reverse transcription) 本论文通过向细胞转染干扰载体下调LINE-1ORF1来验证以前通过基因芯片筛选出的差异表达基因,我们对显著性差异基因结构分析发现,部分基因的上游或者下游具有LINE-1高度同源的序列,提示LINE-1对其调控可能与这些序列的特征有关,另外LINE-15'UTR含有双向启动子,其激活后可生成双链RNA,继而加工生成siRNA,同时也不排除其单链转录产物加工生成siRNA的可能。基于这些因素,我们拟通过构建含有LINE-15'UTR正向和反向序列的重组表达载体,以及LINE-1ORF1干扰表达载体,转染HepG2细胞,探寻LINE-1对候选靶基因调控的机制。
[Abstract]:In recent years, it has been found that many repeat sequences play an important role in the regulation of gene expression in eukaryotic genome. A large part of non-coding sequences play an important role in the transcription, translation and expression of coding genes. The activation of these moveable elements can change the expression of adjacent genes, and the new insertion sites in or near the coding region can cause mutations and cause changes in gene expression and genome remodeling. In these repeats, there is a retrotransposon LINE-1, that is widely distributed in the genome. LINE-1 is the dominant transposon in the human genome. In normal differentiated cells, the promoter of LINE-1 is highly methylated. It was silenced, but the germ cells developed, the embryo developed early and the tumor cells were activated. It has been shown that LINE-1 is active in almost all tumor cells, suggesting that its activation may be involved in the common mechanism of tumorigenesis. Therefore, the study of LINE-1 is of great significance in understanding the molecular mechanism of tumorigenesis, development and regulation, as well as the target of tumor drugs. At the same time, it also provides a new target for the development of new tumor therapy drugs. The full-length LINE-1 gene includes two open reading frames, ORF1 and ORF2, which express two proteins, ORFlp and ORF2p,. The expression of ORFlp and ORF2p, is much higher than that of ORF2p, ORF1p. Among them, ORFlp has nucleic acid binding activity with its own nucleic acid, forming nucleic acid protein complex (RNP), while ORF2p has kernel acid enzyme and exonuclease activity, and performs reverse locus function together with ORF1p. The mechanism is TPRT (Target-primed reverse transcription). In this paper, the differentially expressed genes which were screened by gene chip were verified by down-regulation of LINE-1ORF1 by transfection of interference vector into cells. We found that significant differentially expressed genes were obtained by structural analysis of significant differentially expressed genes. Some genes have highly homologous LINE-1 sequences upstream or downstream, suggesting that the regulation of LINE-1 on these sequences may be related to the characteristics of these sequences. In addition, LINE-15'UTR contains bidirectional promoters, which can generate double-stranded RNA, after activation. Furthermore, the production of siRNA, by processing does not rule out the possibility of producing siRNA by processing its single strand transcription product. Based on these factors, we intend to construct recombinant expression vectors containing LINE-15'UTR forward and reverse sequences and LINE-1ORF1 interference expression vectors to transfect HepG2 cells and explore the mechanism of LINE-1 regulation on candidate target genes.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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