人BMPR2基因5’侧翼序列的结构与功能研究

发布时间：2018-02-22 11:57

本文关键词： II型骨形成蛋白受体基因转录起始位点 5　出处：《暨南大学》2007年博士论文　论文类型：学位论文

【摘要】： BMPR2基因为Ⅱ型骨形成蛋白受体(BMPR-Ⅱ)的编码基因。BMPR-Ⅱ属于TGF-β受体超家族成员之一，为传导BMP信号所必需。在胚胎发育和成熟个体中，BMP参与调控多种类型的细胞和组织的增殖、凋亡、分化，以及参与趋化、血管生成、基质产生等过程。近年来，BMPR2基因突变致肺动脉高压是一个研究的热点；另外，BMP信号通路由于其与肿瘤的发生之间的密切关系也备受关注。作为一种多功能的基因，其表达理应受到精确调控，因而揭示其表达调控规律对于阐明其正常的生理功能和异常的病理机制无疑具有极其重要的意义。本研究，我们分离人BMPR2基因的5′侧翼区，分析其结构特点，预测和寻找参与该基因表达调控的重要区域和元件。具体研究情况及结果如下： 1．在生物信息学预测转录起始位点及RT-PCR步移前推cDNA 5′端的基础上，运用SMART RACE技术成功地确定了人BMPR2基因的转录起始位点，它在翻译起始密码子上游1013nt处。 2．对人BMPR2基因非同寻常长的5′-前导序列(1013nt)进行了生物信息学分析，发现其GC含量高达62％，预测其会形成严重的二级结构从而影响翻译的效率；由于该区域缺乏典型的内部核糖体进入位点(IRES)，可能不存在帽不依赖的翻译起始；按照翻译起始的扫描学说，推测该区域中存在的6个具有中等强度翻译起始能力的上游开放读码框(uORF)对下游真正的开放读码框的翻译具有负调控作用。 3．以人外周血基因组DNA为模板，PCR扩增构建了包含人BMPR2基因5′侧翼近5.5Kb序列的重组质粒pGL3-Basic-5.5K，用于测序及后续实验。 4．以pGL3-Basic-5.5K为模板，构建了6个不同片断长度的缺失突变体，分别在Hela细胞、肺腺癌细胞A549和肝癌细胞SMMC7721中用promega的双萤光素酶报告实验系统检测其启动子活性。结果显示-1569bp～-1229bp(注：翻译起始密码子ATG中的A定为+1)序列有较密集的潜在转录因子结合位点，可能含有重要的转录激活元件，为BMPR2基因的关键启动区；另外还提示-2333bp～-1570bp和-5106bp～--2875bp区段含有较强的转录负调控元件，而-2874bp～-2334bp区段含有较强的转录正调控元件。 5．将跨越-5106bp～--2875bp区段的DNA序列与上述实验得到的人BMPR2基因的关键启动序列通过重叠延伸PCR连成嵌合片段，克隆到pGL3-Basic载体上构建重组报告质粒进行萤光素酶实验，证实-5106bp～--2875bp区段确实有很强的转录抑制作用，这种抑制作用可能与4个Alu元件有关。 6．人BMPR2基因转录起始位点附近有几个潜在的SP1结合位点，提示该基因可能受SP1激活，为此我们构建了SP1真核表达载体pcDNA3.1(+)-SPI，与人BMPR2基因启动子-萤光素酶基因报告质粒共转Hela细胞，结果表明SP1确实能激活人BMPR2基因的启动子，且具有剂量依赖性。进一步突变BMPR2启动子序列内的其中2个潜在SP1结合位点能显著降低启动子活性，而且EMSA实验表明这2个位点均有与SP1特异结合的能力，为SP1反应元件。 7．人BMPR2基因转录起始位点下游+50nt处有一GGC三核苷酸重复，检测了150份正常人的DNA样品，发现了三种基因型：GGC12／GGC12(148例)，GGC12／GGC11(1例)和GGC12／GGC8(1例)。等位基因GGC12的频率为99.33％，，而GGC11、GGC8的频率均为0.33％。 8．构建包含人BMPR2基因核心启动序列及5′前导序列中GGC12重复序列发生定点突变的突变体，萤光素酶报告基因表达检测显示该突变体的转录活性明显下降，提示该重复序列对人BMPR2基因的表达具有正调控作用。 9．人BMPR2基因GGC三核苷酸重复及周边序列处在一个CpG岛中，甲基化特异PCR(MSP)检测显示人胚肺成纤维细胞HELF的CpG岛高甲基化而巨细胞肺癌细胞PGCL3中该CpG岛非甲基化；同时荧光定量PCR结果证实HELF细胞中的BMPR2基因的转录水平明显低于PGCL3，提示CpG岛的甲基化可能参与了BMPR2基因的转录调控。
[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.

【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R346

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