L1逆转座子编码序列ORF1的功能研究

发布时间：2018-01-12 03:24

本文关键词：L1逆转座子编码序列ORF1的功能研究　出处：《中国人民解放军军事医学科学院》2011年硕士论文　论文类型：学位论文

更多相关文章： LINE-1 ORF1 细胞增殖 基因表达调控

【摘要】：近来研究显示,在基因组中过去认为“垃圾序列”的一些重复序列,可能具有重要的功能。这些重复序列中,大部分是移动元件,包括转座子(transposon)与逆转座子(retrotransposon),其中逆转座子分为含有末端重复序列(LTR,long terminal repeats)与非含有末端重复序列两类,后者为SINEs(short interspersed nuclear elements,短散布重复序列)和LINEs(long interspersed nuclear elements,长散布重复序列)。在LINEs中LINE-1是其主要组成成员,在基因组中,绝大部分LINE-1是以5’截短的形式存在,全长基因包括两个开放阅读框ORF1和ORF2,表达两种蛋白ORF-1p和ORF-2p,相对于ORF-2p,ORF-1p的表达大大过量。其中ORF-1p具有与自身核酸结合活性,形成核酸蛋白复合物(RNP),而ORF-2p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)。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L1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L1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。研究发现,ORF-1p与目前所有已知蛋白均缺乏同源性,同时其表达量又远过量于与其共同参与转座的ORF-2p,提示其在肿瘤细胞中可能具有重要的作用。本研究对其进行了有关探索。主要表现一下几个方面: (1)ORF-1p过表达对细胞生物学特征的影响: 在此过程中,我们分别构建含有人L1-ORF1p完整、突变体序列重组表达载体以及小鼠ML1-ORF1p重组表达载体,转染细胞后进行RT-PCR、Western Blot以及流式细胞术方法检测,为进一步研究L1-ORF1蛋白及基因的功能提供了基础。结果显示,人与小鼠ORF1p完整蛋白均能显著促进细胞增殖,在人L1-ORF1p不同突变体中,位于第109氨基酸处终止突变的全长序列也能促进细胞增殖,随后对含有109氨基酸的基因进行表达发现,其表达产物不能促进细胞增殖,另外其它全长多处点突变的ORF1p也不能促进细胞增殖,多处突变会造成蛋白高级结构的改变,从而影响其功能的执行,同时非全长蛋白在其全长RNA的帮助下具有全长蛋白功能,说明ORF1在基因水平影响细胞增殖。随后对转染细胞的其它细胞生物学特征进行了鉴定,发现小鼠ORF1p的表达能显著减少NIH3T3细胞周期S期,促进小鼠NIH3T3细胞的细胞周期运转。人ORF1p的天然以及ORF1p109突变的全长构建体表达产物能够使HEK293细胞的S期减少,加快细胞周期运转,而全长突变蛋白ORF1pm、ORF1p48以及ORF1p109e则对细胞周期的S期则没有显著的影响,此结果与细胞增殖实验结果一致。在正常细胞中过表达L1-ORF1p可促进细胞增殖,加快细胞周期,在109氨基酸处带有终止密码的全长基因突变体具有相同的作用,而终止密码位于其它部位的突变体则没有相似的作用,以上提示L1-ORF1的功能可能在RNA水平上起作用,而且与RNA中特异的序列有关。 (2)5’端不同长度截短的L1-ORF1序列对相邻基因的影响: 在此过程中,我们构建了L1-ORF1基因插入到报告基因上下游的报告基因表达载体,由于在天然存在的状态下L1常存在5’端缺失,所以本研究选取三种不同长度的L1-ORF1片段,分别是全长片段、2/3全长片段(5’端截短1/3)、1/3全长片段(5’端截短2/3)。通过设计引物插入酶切位点将人和小鼠的三种不同片段分别插入到pCBG99-control报告基因表达载体中萤光素酶报告基因的上游和下游。为进一步检测L1-ORF1基因对相邻基因表达影响提供了条件。结果显示,L1-ORF1基因片段在报告基因上游和下游都能抑制报告基因的表达,而且基因片段的不同片段(5’端截短)对报告基因的表达也有不同的影响。在插入报告基因上游,L1-ORF1基因片段对报告基因影响最为显著的是只含3’端1/3的小片段,其可能原因是此区域位于基因保守区含有特定结构域;在插入报告基因下游,L1-ORF1基因片段对报告基因的影响随片段长度增加而加强。 (3)L1-ORF1序列对相邻基因表达影响的机制: 在此过程中,我们对细胞进行甲基化抑制处理,结果显示在甲基转移酶受到抑制的情况下,L1-ORF1基因对萤光素酶报告基因的抑制作用有明显的降低,说明L1-ORF1基因的相邻基因起抑制作用有可能通过甲基化作用而实现,表明其作用机制可能是表观遗传学调控的结果。综上说明,L1-ORF1的表达能通过顺式和反式调控作用促进细胞生长,其在肿瘤的发生与发展过程中具有重要的作用,并可能作为肿瘤药物治疗的新靶点。本研究初步探讨了L1-ORF1蛋白和基因的功能,为进一步研究其作用机制奠定了基础。
[Abstract]:Recent studies show that in the past that "some of the genome sequence repeat sequence of garbage", may have important functions. These repeats, most of mobile elements, including transposons and retrotransposons (transposon) (retrotransposon), which is divided into sub block reverse containing terminal repeats (LTR long, terminal repeats) at the end of two repeats and non containing, which is SINEs (short interspersed nuclear elements, short interspersed repetitive sequences (long) and LINEs interspersed nuclear elements, long interspersed repeats). The LINEs LINE-1 is the major member, in the genome, most of the LINE-1 is the existence of 5 "truncated form, full length the gene consists of two open reading frames ORF1 and ORF2, the expression of two proteins ORF-1p and ORF-2p, compared with ORF-2p, the expression of ORF-1p and ORF-1p is greatly excessive. Combined with its nucleic acid The activity of the formation of protein nucleic acid complexes (RNP), and ORF-2p with endonuclease and exonuclease activity, together with the ORF1p implementation of retrotransposition function, the mechanism of TPRT (Target-primed reverse transcription).
In normal differentiated cells, the LINE-1 promoter is highly methylated, caused by the silence, but the development of germ cells, is activated in early embryonic development and tumor cells. Studies show that in almost all tumor cells in L1 were activated, suggesting that its activation may involve common mechanisms of tumorigenesis so the research on L1. Through the understanding of tumorigenesis, plays an important role in the molecular mechanism of development and regulation of the tumor and drug targets, but also provide a new target for the development of new drugs for the treatment of cancer. The study found that ORF-1p and all known proteins were lack of homology, while its expression is far in excess of participate in the transposition of ORF-2p, suggesting that it may play an important role in tumor cells. This study was carried out on the exploration of the following aspects mainly:
(1) the effect of overexpression of ORF-1p on cell biological characteristics:
In this process, we constructed the human L1-ORF1p mutant sequence integrity, recombinant expression vector and recombinant mouse ML1-ORF1p cells transfected by RT-PCR, Western, Blot and flow cytometry detection method, provide the basis for further study of L1-ORF1 protein and gene function. The results show that human and mouse ORF1p protein can complete significantly promote cell proliferation in human L1-ORF1p mutants, located 109th amino acids at the termination sequence mutation can promote cell proliferation, then the gene contains 109 amino acid found in the expression, the expression product can promote cell proliferation and other full-length multiple point mutations of ORF1p can promote cell proliferation and multiple the mutation causes protein tertiary structure change, thus affecting the execution of the function, while the non full-length protein in its full-length RNA help out A full-length protein, indicating that ORF1 gene level affect cell proliferation. Then other cell biological characteristics of the transfected cells were identified, found that the expression of murine ORF1p can significantly decrease the NIH3T3 S phase of the cell cycle, promotes cell cycle of mouse NIH3T3 cell operation. ORF1p and natural mutation constructs full-length ORF1p109 gene product can the S phase of HEK293 cells reduced, accelerate cell cycle progression, and the full-length mutant protein ORF1pm, ORF1p48 and ORF1p109e on the cell cycle of S phase had no significant effect, the results of cell proliferation and the experimental results are consistent.
Overexpression of L1-ORF1p can promote cell proliferation in normal cells, accelerate cell cycle in 109 amino acid with the full-length gene stop codon mutants have the same effect, and in other parts of the stop codon mutants were not similar, suggesting that L1-ORF1 might play a role in the RNA level, but also related to the sequence of RNA specific.
(2) the effect of the 5 'end L1-ORF1 sequences with different lengths on adjacent genes:
In this process, we constructed the L1-ORF1 gene inserted into reporter gene downstream reporter gene expression vector, due to the natural state of existence under the 5 'end of L1 is often missing, so this study selected three different length of L1-ORF1 fragments were full-length fragments, the full length 2/3 fragment (5' end truncated 1/3) 1/3, full length fragment (5 'end truncated 2/3). Three different primers inserted fragment by restriction sites of human and mouse were inserted into the pCBG99-control gene expression vector in the upstream and downstream of luciferase. Provides conditions for further detection of L1-ORF1 gene expression of adjacent genes. The results showed that L1-ORF1 gene can inhibit the expression of the reporter gene in the reporter gene upstream and downstream, and different fragments of gene fragments (5' end truncated) on the expression of the reporter gene have different effects. In the plug The reporter gene upstream influence L1-ORF1 gene fragment of the reporter gene was most marked with only 3 'end of 1/3 fragments, the possible reason is this area is located in the conserved regions of the gene containing a specific domain; the inserted into the downstream reporter gene, L1-ORF1 gene fragment of the reporter gene influence with the fragment length increased.
(3) the mechanism of the effect of L1-ORF1 sequence on the expression of adjacent genes:
In this process, we suppress methylation in cells, results showed that methyl inhibited transferase in case of inhibitory effects of L1-ORF1 gene on luciferase reporter gene significantly reduced, indicating the adjacent L1-ORF1 gene may inhibit by methylation, suggesting that its mechanism of action may is the result of epigenetic regulation. In conclusion, the expression of L1-ORF1 by CIS and trans regulatory role in promoting cell growth, which plays an important role in the occurrence and development of tumor, and may serve as a new target of tumor therapy. This study explored the L1-ORF1 protein and gene function. Laid the foundation for the further study of its mechanism.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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