發(fā)布時(shí)間：2018-02-22 11:57

  本文關(guān)鍵詞： II型骨形成蛋白受體基因 轉(zhuǎn)錄起始位點(diǎn) 5　出處：《暨南大學(xué)》2007年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】： BMPR2基因?yàn)棰蛐凸切纬傻鞍资荏w(BMPR-Ⅱ)的編碼基因。BMPR-Ⅱ?qū)儆赥GF-β受體超家族成員之一，為傳導(dǎo)BMP信號(hào)所必需。在胚胎發(fā)育和成熟個(gè)體中，BMP參與調(diào)控多種類(lèi)型的細(xì)胞和組織的增殖、凋亡、分化，以及參與趨化、血管生成、基質(zhì)產(chǎn)生等過(guò)程。近年來(lái)，BMPR2基因突變致肺動(dòng)脈高壓是一個(gè)研究的熱點(diǎn)；另外，BMP信號(hào)通路由于其與腫瘤的發(fā)生之間的密切關(guān)系也備受關(guān)注。作為一種多功能的基因，其表達(dá)理應(yīng)受到精確調(diào)控，因而揭示其表達(dá)調(diào)控規(guī)律對(duì)于闡明其正常的生理功能和異常的病理機(jī)制無(wú)疑具有極其重要的意義。本研究，我們分離人BMPR2基因的5′側(cè)翼區(qū)，分析其結(jié)構(gòu)特點(diǎn)，預(yù)測(cè)和尋找參與該基因表達(dá)調(diào)控的重要區(qū)域和元件。具體研究情況及結(jié)果如下： 1．在生物信息學(xué)預(yù)測(cè)轉(zhuǎn)錄起始位點(diǎn)及RT-PCR步移前推cDNA 5′端的基礎(chǔ)上，運(yùn)用SMART RACE技術(shù)成功地確定了人BMPR2基因的轉(zhuǎn)錄起始位點(diǎn)，它在翻譯起始密碼子上游1013nt處。 2．對(duì)人BMPR2基因非同尋常長(zhǎng)的5′-前導(dǎo)序列(1013nt)進(jìn)行了生物信息學(xué)分析，發(fā)現(xiàn)其GC含量高達(dá)62％，預(yù)測(cè)其會(huì)形成嚴(yán)重的二級(jí)結(jié)構(gòu)從而影響翻譯的效率；由于該區(qū)域缺乏典型的內(nèi)部核糖體進(jìn)入位點(diǎn)(IRES)，可能不存在帽不依賴的翻譯起始；按照翻譯起始的掃描學(xué)說(shuō)，推測(cè)該區(qū)域中存在的6個(gè)具有中等強(qiáng)度翻譯起始能力的上游開(kāi)放讀碼框(uORF)對(duì)下游真正的開(kāi)放讀碼框的翻譯具有負(fù)調(diào)控作用。 3．以人外周血基因組DNA為模板，PCR擴(kuò)增構(gòu)建了包含人BMPR2基因5′側(cè)翼近5.5Kb序列的重組質(zhì)粒pGL3-Basic-5.5K，用于測(cè)序及后續(xù)實(shí)驗(yàn)。 4．以pGL3-Basic-5.5K為模板，構(gòu)建了6個(gè)不同片斷長(zhǎng)度的缺失突變體，分別在Hela細(xì)胞、肺腺癌細(xì)胞A549和肝癌細(xì)胞SMMC7721中用promega的雙螢光素酶報(bào)告實(shí)驗(yàn)系統(tǒng)檢測(cè)其啟動(dòng)子活性。結(jié)果顯示-1569bp～-1229bp(注：翻譯起始密碼子ATG中的A定為+1)序列有較密集的潛在轉(zhuǎn)錄因子結(jié)合位點(diǎn)，可能含有重要的轉(zhuǎn)錄激活元件，為BMPR2基因的關(guān)鍵啟動(dòng)區(qū)；另外還提示-2333bp～-1570bp和-5106bp～--2875bp區(qū)段含有較強(qiáng)的轉(zhuǎn)錄負(fù)調(diào)控元件，而-2874bp～-2334bp區(qū)段含有較強(qiáng)的轉(zhuǎn)錄正調(diào)控元件。 5．將跨越-5106bp～--2875bp區(qū)段的DNA序列與上述實(shí)驗(yàn)得到的人BMPR2基因的關(guān)鍵啟動(dòng)序列通過(guò)重疊延伸PCR連成嵌合片段，克隆到pGL3-Basic載體上構(gòu)建重組報(bào)告質(zhì)粒進(jìn)行螢光素酶實(shí)驗(yàn)，證實(shí)-5106bp～--2875bp區(qū)段確實(shí)有很強(qiáng)的轉(zhuǎn)錄抑制作用，這種抑制作用可能與4個(gè)Alu元件有關(guān)。 6．人BMPR2基因轉(zhuǎn)錄起始位點(diǎn)附近有幾個(gè)潛在的SP1結(jié)合位點(diǎn)，提示該基因可能受SP1激活，為此我們構(gòu)建了SP1真核表達(dá)載體pcDNA3.1(+)-SPI，與人BMPR2基因啟動(dòng)子-螢光素酶基因報(bào)告質(zhì)粒共轉(zhuǎn)Hela細(xì)胞，結(jié)果表明SP1確實(shí)能激活人BMPR2基因的啟動(dòng)子，且具有劑量依賴性。進(jìn)一步突變BMPR2啟動(dòng)子序列內(nèi)的其中2個(gè)潛在SP1結(jié)合位點(diǎn)能顯著降低啟動(dòng)子活性，而且EMSA實(shí)驗(yàn)表明這2個(gè)位點(diǎn)均有與SP1特異結(jié)合的能力，為SP1反應(yīng)元件。 7．人BMPR2基因轉(zhuǎn)錄起始位點(diǎn)下游+50nt處有一GGC三核苷酸重復(fù)，檢測(cè)了150份正常人的DNA樣品，發(fā)現(xiàn)了三種基因型：GGC12／GGC12(148例)，GGC12／GGC11(1例)和GGC12／GGC8(1例)。等位基因GGC12的頻率為99.33％，，而GGC11、GGC8的頻率均為0.33％。 8．構(gòu)建包含人BMPR2基因核心啟動(dòng)序列及5′前導(dǎo)序列中GGC12重復(fù)序列發(fā)生定點(diǎn)突變的突變體，螢光素酶報(bào)告基因表達(dá)檢測(cè)顯示該突變體的轉(zhuǎn)錄活性明顯下降，提示該重復(fù)序列對(duì)人BMPR2基因的表達(dá)具有正調(diào)控作用。 9．人BMPR2基因GGC三核苷酸重復(fù)及周邊序列處在一個(gè)CpG島中，甲基化特異PCR(MSP)檢測(cè)顯示人胚肺成纖維細(xì)胞HELF的CpG島高甲基化而巨細(xì)胞肺癌細(xì)胞PGCL3中該CpG島非甲基化；同時(shí)熒光定量PCR結(jié)果證實(shí)HELF細(xì)胞中的BMPR2基因的轉(zhuǎn)錄水平明顯低于PGCL3，提示CpG島的甲基化可能參與了BMPR2基因的轉(zhuǎn)錄調(diào)控。
[Abstract]:BMPR2 based because of bone morphogenetic protein receptor type II (BMPR- II) encoding gene.BMPR- II belongs to TGF- beta receptor superfamily, are necessary for the conduction of BMP signal. In the embryonic development and mature individuals, BMP is involved in the regulation of various types of cells and tissue proliferation, apoptosis, differentiation, and chemotaxis. The process of angiogenesis, matrix production. In recent years, BMPR2 gene mutation induced pulmonary hypertension is a focus of research; in addition, due to the close relationship between the BMP pathway and tumor occurrence is also of concern. As a kind of multifunctional gene, its expression should be precisely controlled, thus revealing its expression regulation is of great significance to clarify its normal physiological function and abnormal pathological mechanism. In this study, we isolated the human BMPR2 gene 5 'flanking region, analyzes the structure, forecasting and looking to participate in the Important regions and components of gene expression regulation. Specific research and results are as follows:
1., based on bioinformatics prediction of transcriptional start site and cDNA 5 'end before RT-PCR walking, we successfully identified the transcription start site of human BMPR2 gene based on SMART RACE technology, which is at the upstream of translation initiation codon 1013nt.
2. of the human BMPR2 gene 5 '- extraordinary long preamble sequence (1013nt) by bioinformatics analysis, we found that the GC content reaches 62%, the prediction will form two level structure and serious influence the translation efficiency; in the region because of the lack of typical internal ribosome entry site (IRES), there may be no cap do not rely on the translation initiation; according to the theory of scanning the initiation of translation, there are probably in the region of 6 with moderate intensity of translation initiation ability of the upstream open reading frame (uORF) of the real open reading frame downstream translation has negative regulatory role.
3., using human peripheral blood genomic DNA as template and PCR amplification, we constructed a recombinant plasmid pGL3-Basic-5.5K containing human BMPR2 gene 5 'flanking nearly 5.5Kb sequence, which was used for sequencing and subsequent experiments.
4. pGL3-Basic-5.5K as a template, constructed deletion mutants of 6 different fragment length, respectively, in Hela cells, lung cancer cells A549 and SMMC7721 cells with Promega dual luciferase report system to detect the promoter activity of experiment. The results showed that -1569bp ~ -1229bp (Note: the translation start codon ATG in A as a potential transcription factor +1) sequence dense binding sites may contain important transcription activation elements, the promoter region of BMPR2 gene is the key; in addition -2333bp ~ -1570bp and -5106bp ~ --2875bp section containing transcription strong negative regulatory elements also suggest that the -2874bp ~ -2334bp section contains a strong positive transcriptional regulatory elements.
The key to start the sequence of 5. spans of human BMPR2 gene -5106bp ~ DNA sequence and the experimental section of the --2875bp obtained by PCR into chimeric fragment overlap extension, cloned into pGL3-Basic vector to construct the recombinant plasmid for luciferase report experiments confirmed that -5106bp ~ --2875bp section does have a strong inhibition of transcription, this inhibition may with 4 Alu elements.
There are several potential binding sites of SP1 near 6. BMPR2 gene transcription initiation site, suggesting that the gene may be affected by the activation of SP1, we constructed SP1 eukaryotic expression vector pcDNA3.1 (+) -SPI, and BMPR2 gene promoter luciferase gene reporter plasmid co transfection of Hela cells, the results show that SP1 can activate the human BMPR2 gene promoter in a dose-dependent manner. The mutation of BMPR2 promoter sequence in which 2 potential SP1 binding sites significantly reduced promoter activity, and EMSA experiments show that the ability to combine these 2 loci were specific for SP1 and SP1, the reaction components.
7. human BMPR2 gene transcription start site at +50nt downstream of a GGC trinucleotide repeat, examined 150 normal DNA samples, found three genotypes: GGC12 / GGC12 (148 cases), GGC12 / GGC11 (1 cases) and GGC12 / GGC8 (1 cases). The frequencies of GGC12 allele were 99.33%, GGC11, GGC8 frequency was 0.33%.
8. constructs containing human BMPR2 gene core promoter sequences and GGC12 5 'preamble sequence repeat sequence in site directed mutagenesis of the mutant luciferase reporter gene expression assay showed that the transcriptional activity of the mutant decreased significantly, suggesting that the expression of the repeat sequence of the human BMPR2 gene has a positive regulatory effect.
9. BMPR2 trinucleotide repeats of GGC gene and the flanking sequence in a CpG Island, methylation specific PCR (MSP) detection of human embryonic lung fibroblast HELF hypermethylation of the CpG island and the island of CpG lung cancer cell line PGCL3 in unmethylated display; and fluorescence quantitative PCR showed that the transcription level of BMPR2 gene in HELF cells in the lower than PGCL3, suggesting that CpG island methylation may be involved in the transcriptional regulation of BMPR2 gene.

【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 夏平安,崔保安,梁宏德,王巖,劉興友;真核基因的轉(zhuǎn)錄激活調(diào)控[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2004年04期

2 戴明華,王恩多,謝雍,姜衛(wèi)紅,趙國(guó)屏;青霉素�；�酶活性中心的定點(diǎn)突變[J];生物化學(xué)與生物物理學(xué)報(bào);1999年05期

3 薛麗香,童坦君,張宗玉;報(bào)告基因的選擇及其研究趨向[J];生理科學(xué)進(jìn)展;2002年04期

4 張梅,司履生;重組PCR——一種快速體外基因重組技術(shù)[J];西安醫(yī)科大學(xué)學(xué)報(bào);2001年03期

5 荊志成;;波生坦治療肺動(dòng)脈高壓藥理機(jī)制及其臨床評(píng)價(jià)[J];中國(guó)醫(yī)院用藥評(píng)價(jià)與分析;2005年06期

本文編號(hào)：1524352