本文關(guān)鍵詞：L1逆轉(zhuǎn)座子編碼序列ORF1的功能研究　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： LINE-1 ORF1 細(xì)胞增殖 基因表達(dá)調(diào)控

【摘要】：近來(lái)研究顯示,在基因組中過(guò)去認(rèn)為“垃圾序列”的一些重復(fù)序列,可能具有重要的功能。這些重復(fù)序列中,大部分是移動(dòng)元件,包括轉(zhuǎn)座子(transposon)與逆轉(zhuǎn)座子(retrotransposon),其中逆轉(zhuǎn)座子分為含有末端重復(fù)序列(LTR,long terminal repeats)與非含有末端重復(fù)序列兩類,后者為SINEs(short interspersed nuclear elements,短散布重復(fù)序列)和LINEs(long interspersed nuclear elements,長(zhǎng)散布重復(fù)序列)。在LINEs中LINE-1是其主要組成成員,在基因組中,絕大部分LINE-1是以5’截短的形式存在,全長(zhǎng)基因包括兩個(gè)開(kāi)放閱讀框ORF1和ORF2,表達(dá)兩種蛋白ORF-1p和ORF-2p,相對(duì)于ORF-2p,ORF-1p的表達(dá)大大過(guò)量。其中ORF-1p具有與自身核酸結(jié)合活性,形成核酸蛋白復(fù)合物(RNP),而ORF-2p具有內(nèi)核酸酶及外切酶活性,與ORF1p一起執(zhí)行逆轉(zhuǎn)座功能,其機(jī)制為TPRT(Target-primed reverse transcription)。 在正常分化的細(xì)胞中,LINE-1的啟動(dòng)子為高度甲基化狀態(tài),造成其沉默,但生殖細(xì)胞發(fā)育、胚胎發(fā)育的早期以及腫瘤細(xì)胞中則為激活狀態(tài)。研究表明在幾乎所有的腫瘤細(xì)胞中L1均為激活狀態(tài),提示其激活可能涉及腫瘤發(fā)生的共同機(jī)制。所以通過(guò)L1的研究對(duì)了解腫瘤發(fā)生、發(fā)展和調(diào)控的分子機(jī)理以及腫瘤藥物靶標(biāo)具有重要意義,同時(shí)也為開(kāi)發(fā)新型的腫瘤治療藥物提供新的靶點(diǎn)。研究發(fā)現(xiàn),ORF-1p與目前所有已知蛋白均缺乏同源性,同時(shí)其表達(dá)量又遠(yuǎn)過(guò)量于與其共同參與轉(zhuǎn)座的ORF-2p,提示其在腫瘤細(xì)胞中可能具有重要的作用。本研究對(duì)其進(jìn)行了有關(guān)探索。主要表現(xiàn)一下幾個(gè)方面: (1)ORF-1p過(guò)表達(dá)對(duì)細(xì)胞生物學(xué)特征的影響: 在此過(guò)程中,我們分別構(gòu)建含有人L1-ORF1p完整、突變體序列重組表達(dá)載體以及小鼠ML1-ORF1p重組表達(dá)載體,轉(zhuǎn)染細(xì)胞后進(jìn)行RT-PCR、Western Blot以及流式細(xì)胞術(shù)方法檢測(cè),為進(jìn)一步研究L1-ORF1蛋白及基因的功能提供了基礎(chǔ)。結(jié)果顯示,人與小鼠ORF1p完整蛋白均能顯著促進(jìn)細(xì)胞增殖,在人L1-ORF1p不同突變體中,位于第109氨基酸處終止突變的全長(zhǎng)序列也能促進(jìn)細(xì)胞增殖,隨后對(duì)含有109氨基酸的基因進(jìn)行表達(dá)發(fā)現(xiàn),其表達(dá)產(chǎn)物不能促進(jìn)細(xì)胞增殖,另外其它全長(zhǎng)多處點(diǎn)突變的ORF1p也不能促進(jìn)細(xì)胞增殖,多處突變會(huì)造成蛋白高級(jí)結(jié)構(gòu)的改變,從而影響其功能的執(zhí)行,同時(shí)非全長(zhǎng)蛋白在其全長(zhǎng)RNA的幫助下具有全長(zhǎng)蛋白功能,說(shuō)明ORF1在基因水平影響細(xì)胞增殖。隨后對(duì)轉(zhuǎn)染細(xì)胞的其它細(xì)胞生物學(xué)特征進(jìn)行了鑒定,發(fā)現(xiàn)小鼠ORF1p的表達(dá)能顯著減少NIH3T3細(xì)胞周期S期,促進(jìn)小鼠NIH3T3細(xì)胞的細(xì)胞周期運(yùn)轉(zhuǎn)。人ORF1p的天然以及ORF1p109突變的全長(zhǎng)構(gòu)建體表達(dá)產(chǎn)物能夠使HEK293細(xì)胞的S期減少,加快細(xì)胞周期運(yùn)轉(zhuǎn),而全長(zhǎng)突變蛋白ORF1pm、ORF1p48以及ORF1p109e則對(duì)細(xì)胞周期的S期則沒(méi)有顯著的影響,此結(jié)果與細(xì)胞增殖實(shí)驗(yàn)結(jié)果一致。 在正常細(xì)胞中過(guò)表達(dá)L1-ORF1p可促進(jìn)細(xì)胞增殖,加快細(xì)胞周期,在109氨基酸處帶有終止密碼的全長(zhǎng)基因突變體具有相同的作用,而終止密碼位于其它部位的突變體則沒(méi)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,而且與RNA中特異的序列有關(guān)。 (2)5’端不同長(zhǎng)度截短的L1-ORF1序列對(duì)相鄰基因的影響: 在此過(guò)程中,我們構(gòu)建了L1-ORF1基因插入到報(bào)告基因上下游的報(bào)告基因表達(dá)載體,由于在天然存在的狀態(tài)下L1常存在5’端缺失,所以本研究選取三種不同長(zhǎng)度的L1-ORF1片段,分別是全長(zhǎng)片段、2/3全長(zhǎng)片段(5’端截短1/3)、1/3全長(zhǎng)片段(5’端截短2/3)。通過(guò)設(shè)計(jì)引物插入酶切位點(diǎn)將人和小鼠的三種不同片段分別插入到pCBG99-control報(bào)告基因表達(dá)載體中螢光素酶報(bào)告基因的上游和下游。為進(jìn)一步檢測(cè)L1-ORF1基因?qū)ο噜徎虮磉_(dá)影響提供了條件。結(jié)果顯示,L1-ORF1基因片段在報(bào)告基因上游和下游都能抑制報(bào)告基因的表達(dá),而且基因片段的不同片段(5’端截短)對(duì)報(bào)告基因的表達(dá)也有不同的影響。在插入報(bào)告基因上游,L1-ORF1基因片段對(duì)報(bào)告基因影響最為顯著的是只含3’端1/3的小片段,其可能原因是此區(qū)域位于基因保守區(qū)含有特定結(jié)構(gòu)域;在插入報(bào)告基因下游,L1-ORF1基因片段對(duì)報(bào)告基因的影響隨片段長(zhǎng)度增加而加強(qiáng)。 (3)L1-ORF1序列對(duì)相鄰基因表達(dá)影響的機(jī)制: 在此過(guò)程中,我們對(duì)細(xì)胞進(jìn)行甲基化抑制處理,結(jié)果顯示在甲基轉(zhuǎn)移酶受到抑制的情況下,L1-ORF1基因?qū)ξ灩馑孛笀?bào)告基因的抑制作用有明顯的降低,說(shuō)明L1-ORF1基因的相鄰基因起抑制作用有可能通過(guò)甲基化作用而實(shí)現(xiàn),表明其作用機(jī)制可能是表觀遺傳學(xué)調(diào)控的結(jié)果。綜上說(shuō)明,L1-ORF1的表達(dá)能通過(guò)順式和反式調(diào)控作用促進(jìn)細(xì)胞生長(zhǎng),其在腫瘤的發(fā)生與發(fā)展過(guò)程中具有重要的作用,并可能作為腫瘤藥物治療的新靶點(diǎn)。本研究初步探討了L1-ORF1蛋白和基因的功能,為進(jìn)一步研究其作用機(jī)制奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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相關(guān)期刊論文 前2條

1 高旭東;胡明明;朱運(yùn)峰;;下調(diào)LINE-1編碼蛋白ORF-1p對(duì)肺癌細(xì)胞A549生物學(xué)特征的影響[J];中國(guó)生物工程雜志;2010年06期

2 胡明明;朱運(yùn)峰;王越;王宇;張亮;高旭東;董金凱;于繼云;;LINE-1編碼蛋白L1-ORF1的原核表達(dá)純化和多克隆抗體制備[J];中國(guó)生物工程雜志;2010年10期

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本文編號(hào)：1412487