ORMDL3在哮喘发生中的作用及其转录调控机制研究

发布时间：2018-08-13 08:29

【摘要】：哮喘是一种由环境和遗传因素共同作用导致的复杂疾病,其遗传率约为36%-79%。2007年,对哮喘进行的第一个全基因组关联分析发现了一个新的哮喘易感基因----ORMDL3。研究者发现ORMDL3所在的17q21区域中的遗传多态位点与儿童哮喘显著相关,并且在EB病毒感染的哮喘儿童的淋巴母细胞系中,该区域中的遗传多态还与ORMDL3基因的表达水平密切相关。 ORMDL3基因位于17q21,是ORM基因家族的一员。在酵母中,该家族含有ORMDL1和ORMDL2两个成员,而在人类中还有ORMDL3,共三个家族成员。在酵母中,该基因家族与神经鞘磷脂代谢平衡相关,是神经鞘磷脂合成过程中第一个限速酶的负调控者,提示神经鞘磷脂代谢可能在哮喘的发生过程中具有重要作用。另外,在酵母中敲除ORM基因,表现为生长缓慢和对有毒物质的敏感性增强,这一现象在转染入人类ORMDL3基因的mRNA后得以拯救。ORMDL3基因编码一个含有153个氨基酸的内质网四次跨膜蛋白,其在人类组织中表达广泛,特别是在肝脏和外周血淋巴细胞中高表达。研究表明ORMDL3在内质网上与钙泵共定位,抑制钙泵的功能,影响钙离子平衡并且能够促进内质网应激和未折叠蛋白反应,表明ORMDL3可能参与内质网有关的炎症反应。另外,人们还发现在儿童脐带血单核细胞中17q21区域中的遗传多态不但与ORMDL3, GSDMA基因的表达水平相关,而且还与IL-17的分泌相关,表明该区域可能在免疫系统成熟的早期阶段与T细胞的发育分化有关。而除了与哮喘相关之外,人们还发现ORMDL3与克隆恩病、强直性脊柱炎、系统性红斑狼疮、Ⅰ型糖尿病、神经胶质瘤、原发性胆汁肝硬化、过敏性鼻炎等多种炎症性疾病显著相关。自从发现ORMDL3是一个新的哮喘易感基因,在很短的时间内,人们又在多个不同种族人群中进行了验证,结果发现ORMDL3基因在不同人群中均与儿童哮喘显著相关。但是所有这些研究都是在儿童哮喘中进行的,那么,ORMDL3基因是否也与成人哮喘相关呢?在EB病毒感染的哮喘儿童淋巴母细胞系中17q21上的遗传多态与ORMDL3基因的表达相关,那么在成人哮喘患者的外周血淋巴细胞中这一现象是否仍然存在呢?哮喘患者体内ORMDL3基因的表达异常是怎样引起的呢?ORMDL3基因的表达调控又是怎样的呢?带着这些问题,本论文进行了以下四个方面的研究：第一部分17q21区域与中国汉族成人哮喘的关联分析为了明确ORMDL3基因所在的17q21区域与成人哮喘之间的相关性,我们从山东大学齐鲁医院呼吸科收集了710例无亲缘关系的成人哮喘患者,以及从健康体检中心随机收集了656例无亲缘关系、无哮喘病及其它呼吸道疾病及过敏病史的健康体检者作为对照组。根据相关报道及中国人群中该区域的连锁不平衡情况,我们从17q21区域选取了5个单核苷酸多态位点(SNP):rs7216389, rs12603332, rs12936231, rs9303277, rs11557467进行基于群体的病例对照关联分析。结果发现这5个SNP多态位点均与成人哮喘显著相关(P0.05),其中两个SNP多态位点rs9303277, rs11557467与成人哮喘的相关性最强(P0.001)。带有rs11557467位点G等位基因的个体或者rs9303277位点C等位基因的个体的哮喘患病风险显著升高(OR=1.27,95%CI1.07-1.51, P=0.006以及OR=1.27,95%CI1.07-1.49,P=0.005)。即使是在去除性别和年龄的影响以后,这种相关性仍然存在。此外,单体型分析发现,与单体型TCCCG相比,单体型CTGTT且有保护效应,OR=0.81,95%CI0.67-0.97, P=0.02。因此,本部分研究结果表明：ORMDL3基因所在的17q21区域中的遗传多态在中国汉族人群中与成人哮喘显著相关。第二部分17q21区域导致哮喘易感的功能SNP的鉴定由于在Ensembl数据库中显示ORMDL3基因具有2种转录本形式：ENST00000304046(ORMDL3-001)以及ENST00000394169(ORMDL3-002),并且这两个转录本的编码区域是完全相同的,不同的只是非翻译区,因此它们所编码的蛋白质是完全一样的。那么在人体中,ORMDL3基因是以哪一种转录本形式存在呢?另外,Moffatt等人的研究发现,在EB病毒感染的哮喘儿童的淋巴母细胞系中17q21区域中的遗传多态与ORMDL3基因的表达水平密切相关,因而提示遗传多态调节ORMDL3基因表达的异常很可能在哮喘的发生中起着一定的作用。但是到目前为止,已经发现的该区域中与哮喘相关的SNP中没有一个是位于ORMDL3基因的编码区,而我们第一部分选取的SNP位点rs12603332恰好位于该基因的第一内含子中,而已有报道提示位于内含子中的遗传多态可能会影响特定细胞或组织中基因的转录剪接。因此,我们首先检测了ORMDL3基因在人体不同组织中所使用的转录本形式以及该基因第一内含子中的一个SNP位点rs12603332的不同等位基因是否影响ORMDL3基因mRNA的剪接。我们选取rs12603332位点基因型为CC及TT的个体各5例,同时选取人体肝脏组织,食管癌组织,大肠癌组织以及肺组织各12例,分别提取DNA和RNA。另外,根据两个转录本的剪接特点设计引物,用PCR扩增的方法进行检测：一对引物能同时扩增出ORMDL3的两种转录本形式,用以确定ORMDL3基因是否表达同时监控cDNA的质量,另一对引物只能扩增出第二转录本,用于检测第二转录本是否存在。结果显示,在人体外周血淋巴细胞、肝脏组织、食管癌组织、大肠癌组织以及肺组织中都只表达ORMDL3基因的第一转录本形式：ENST00000304046(ORMDL3-001),并且不论是CC基因型还是TT基因型(rs12603332位点)的个体都只表达第一转录本。因此,rs12603332位点并不影响ORMDL3基因的剪接,那么,该区域中的遗传多态导致哮喘的机制是什么呢?哪个位点才是真正的功能位点呢? 由于基因转录水平的改变是介导哮喘或其他疾病易感的一个非常重要的机制,并且基因转录丰度很可能受到某些改变转录因子结合的SNP多态位点的直接影响。因此,我们检测了哮喘患者和正常对照中ORMDL3基因和GSDMB基因的表达情况。从山东大学齐鲁医院呼吸科收集了61例确诊的成人哮喘患者以及70例正常体检个体的新鲜外周血,提取DNA及RNA,运用实时定量PCR的方法进行等位基因差异表达分析。结果发现,哮喘患者外周血淋巴细胞中ORMDL3基因以及GSDMB基因的表达水平显著高于正常对照个体,P0.05,并且携带有rs7216389,rs12603332以及rs12936231位点危险等位基因的个体,其ORMDL3和GSDMB基因的表达水平显著升高,P值均小于0.05。因此,本部分研究结果表明：首先,在人体外周血淋巴细胞、肝脏组织、食管癌组织、大肠癌组织以及肺组织中,ORMDL3基因以第一转录本的形式存在,并且该基因第一内含子中rs12603332位点的不同等位基因并不影响ORMDL3基因mRNA的剪接。其次,成人哮喘患者外周血淋巴细胞中ORMDL3及GSDMB基因的表达水平明显高于正常对照,并且17q21区域中与哮喘相关的遗传多态的危险等位基因能显著升高这两个基因的表达水平。因此,ORMDL3及GSDMB基因的表达异常很可能是该区域诱发哮喘的潜在机制。但由于17q21区域是一个强连锁不平衡的区域,从中筛选出具体的功能位点尚有待于更多的研究来证实。第三部分ORMDL3基因基本启动子的鉴定第二部分研究结果表明,ORMDL3基因的表达异常很可能是导致哮喘发生的原因之一,而关于该基因的转录调控机制目前尚不明确,因此在本部分中我们对该基因的基本启动子进行了分析。首先,我们克隆了ORMDL3基因转录起始点至上游约1.5kb的区域,连入pGL3basic载体中,用双荧光素酶报告基因系统分析其启动子活性。此后运用系列截短的办法寻找包含ORMDL3基因基本启动子的最小区域。结果发现,当我们把序列从-68bp截短到-62bp时,荧光素酶活性迅速下降了70%到80%,表明影响ORMDL3基因启动子活性的反应元件应该位于-68bp到-62bp之间。为了明确哪个转录因子结合在这一区域中影响ORMDL3基因的启动子活性,我们用转录因子预测软件TESS进行分析,结果发现在转录起始点上游-64bp到-56bp区域之间存在一个信号转导及激活转录因子6(STAT6)的结合位点。EMSA及super shift实验表明,在体外情况下STAT6能结合在ORMDL3基因的启动子上。同时ChIP实验也表明,在体内情况下STAT6也能结合在ORMDL3基因的启动子上。本部分实验结果表明,ORMDL3基因的基本启动子区域位于转录起始点上游-68bp以内,并且转录起始点上游-64bp到-56bp区域之间的一个信号转导及激活转录因子6(STAT6)的结合位点对ORMDL3基因的基本启动子活性起主要作用。第四部分STAT6对ORMDL3基因的表达调控作用既然ORMDL3基因启动子区域中的STAT6结合位点对ORMDL3基因的基本启动子活性起主要作用,那么STAT6对ORMDL3基因的表达调控作用具体是怎样的呢?为此,我们构建了STAT6的表达载体,在Jurkat细胞中分别转染STAT6的表达载体或者STAT6的siRNA,结果发现STAT6的高表达或者低表达能显著转录激活或者转录抑制ORMDL3基因基本启动子的荧光素酶报告基因活性及内源性ORMDL3基因mRNA的表达。由于STAT6是一个受细胞因子IL-4/IL-13调控的转录因子,那么ORMDL3基因的表达是否也受IL-4/IL-13的调控呢?因此,我们还检测了IL-4/IL-13对ORMDL3基因表达的影响,结果发现IL-4/IL-13处理能显著激活ORMDL3基因启动子的荧光素酶报告基因活性及内源性ORMDL3基因mRNA的表达,而一旦干扰了STAT6,这一现象就会消失,表明IL-4/IL-13对ORMDL3基因的表达调控作用依赖于STAT6。另外,有研究发现p300也能结合在ORMDL3基因的启动子上,并且它的结合位点与我们发现的STAT6结合位点基本一致。因此,我们进行了ChIP实验,结果发现p300确实也能结合在ORMDL3基因的启动子上。于是我们又进一步进行了IP实验和ChIP-Re-ChIP实验,结果发现p300和STAT6能相互结合,并且它们是以复合物的形式结合在ORMDL3基因的启动子上。事实上,p300是一个协同转录因子,它并不直接结合在DNA序列上,而是通过与其他序列特异的转录因子结合而被招募到启动子上。因此,本部分研究结果表明,转录因子STAT6能转录激活ORMDL3基因的表达,并且IL-4/IL-13处理能显著激活ORMDL3基因的启动子活性及内源性ORMDL3基因mRNA的表达,并且这种激活作用依赖于STAT6。事实上,转录因子STAT6通过招募协同转录因子p300,共同调控ORMDL3基因的表达。这一研究为ORMDL3与炎症及哮喘相关的事实提供了一个合理的解释,同时也为临床上预防和治疗哮喘提供了一个新的途径。
[Abstract]:Asthma is a complex disease caused by environmental and genetic factors. The heritability of asthma is about 36% - 79%. In 2007, the first genome-wide association analysis of asthma revealed a new susceptibility gene, ORMDL3. Researchers found that genetic polymorphisms in the 17q21 region of ORMDL3 were significantly associated with childhood asthma. Furthermore, the genetic polymorphism in this region is also closely related to the expression of ORMDL3 gene in the lymphoblastoid cell lines of asthmatic children infected with EBV.
ORMDL3 gene, located at 17q21, is a member of the ORM gene family. In yeast, ORMDL1 and ORMDL2 are members of the ORMDL3 family, while in humans there are three members of the ORMDL3 family. Transsphingomyelin metabolism may play an important role in the development of asthma. In addition, the knockout of ORM gene in yeast may result in slow growth and increased sensitivity to toxic substances. This phenomenon was saved by transfection of human ORMDL3 gene into the mRNA. ORMDL3 gene encodes an endoplasmic reticulum containing 153 amino acids four times across the membrane. ORMDL3 is widely expressed in human tissues, especially in the liver and peripheral blood lymphocytes. Studies have shown that ORMDL3 co-localizes with calcium pumps in the endoplasmic reticulum, inhibits the function of calcium pumps, affects the balance of calcium ions, and promotes endoplasmic reticulum stress and unfolded protein responses, suggesting that ORMDL3 may be involved in endoplasmic reticulum-related inflammation. In addition, genetic polymorphisms in the 17q21 region of cord blood mononuclear cells in children were found to be associated not only with the expression of ORMDL3 and GSDMA genes, but also with the secretion of IL-17, suggesting that the region may be associated with the development and differentiation of T cells in the early stage of immune system maturation. In addition to asthma, the region is associated with human development. ORMDL3 was also found to be significantly associated with various inflammatory diseases such as Crohn's disease, ankylosing spondylitis, systemic lupus erythematosus, type 1 diabetes mellitus, glioma, primary biliary cirrhosis, and allergic rhinitis.
Since ORMDL3 was found to be a new susceptible gene for asthma, it has been tested in a very short period of time in a number of different ethnic groups. It has been found that ORMDL3 gene is significantly associated with childhood asthma in different populations. What about asthma? The genetic polymorphism of 17q21 in EBV-infected asthmatic children's lymphoblasts is associated with the expression of ORMDL3 gene. Is this still present in the peripheral blood lymphocytes of adult asthmatic patients? How is the abnormal expression of ORMDL3 gene in asthmatic patients caused? With these problems, this paper carries out the following four aspects of research:
Part one association analysis between 17q21 area and asthma in Chinese Han Adults
To clarify the relationship between the 17q21 region of ORMDL3 gene and adult asthma, 710 unrelated adult patients with asthma were collected from the Respiratory Department of Qilu Hospital of Shandong University, and 656 unrelated, non-asthmatic and other respiratory diseases and allergies were randomly collected from the Health Examination Center. Based on the related reports and linkage disequilibrium in the Chinese population, five single nucleotide polymorphisms (SNPs) were selected from the 17q21 region: rs7216389, rs12603332, rs12936231, rs9303277, and rs11557467 for population-based case-control association analysis.
The results showed that the five SNP polymorphisms were significantly associated with adult asthma (P 0.05). Two SNP polymorphisms, rs9303277 and rs11557467, had the strongest association with adult asthma (P 0.001). Individuals with the G allele at rs11557467 or the C allele at rs9303277 had a significantly increased risk of asthma (OR = 1.27, 95% CI 1.0). 7-1.51, P = 0.006 and OR = 1.27, 95% CI 1.07-1.49, P = 0.005). Even after removing the influence of sex and age, this correlation still exists. In addition, haplotype analysis showed that CTGTT had protective effect compared with TCCCG, OR = 0.81, 95% CI 0.67-0.97, P = 0.02.97.
Therefore, the results of this study indicate that the genetic polymorphisms in the 17q21 region of ORMDL3 gene are significantly associated with adult asthma in Chinese Han population.
The second part is the identification of functional SNP in asthma caused by 17q21 region.
Because ORMDL3 gene has two transcript forms in the Ensembl database: ENST00000304046 (ORMDL3-001) and ENST00000394169 (ORMDL3-002), and the coding regions of the two transcripts are identical, but the difference is only the untranslated region, so the protein they encode is identical. In addition, Moffatt et al. found that genetic polymorphism in the 17q21 region of the lymphoblastoid cell line of asthmatic children infected with EBV was closely related to the expression level of ORMDL3 gene, suggesting that the abnormal expression of ORMDL3 gene regulated by genetic polymorphism may be involved in the development of asthma. But so far, none of the asthma-related SNPs in this region is located in the coding region of ORMDL3 gene, and the SNP locus rs12603332 selected in the first part of our study happens to be located in the first intron of the gene. It has been reported that the genetic polymorphism in the intron may be affected. Therefore, we first examined the transcriptional form of ORMDL3 gene used in different tissues of the human body and whether the different alleles of rs12603332, a SNP locus in the first intron of the gene, affected the splicing of ORMDL3 gene mRNA.
DNA and RNA were extracted from human liver, esophageal cancer, colorectal cancer and lung tissues, respectively. In addition, primers were designed according to the splicing characteristics of the two transcripts. PCR amplification was used to detect ORMDL. The results showed that ORMDL3 was only expressed in human peripheral blood lymphocytes, liver tissue, esophageal cancer tissue, colorectal cancer tissue and lung tissue in vitro. The first transcript form of the gene: ENST00000304046 (ORMDL3-001), and individuals of either CC genotype or TT genotype (rs12603332 locus) only express the first transcript. Therefore, rs12603332 locus does not affect the splicing of ORMDL3 gene, so what is the mechanism of genetic polymorphism in this region leading to asthma? Is it a real functional site?
Because changes in gene transcription levels are a very important mechanism mediating susceptibility to asthma or other diseases, and gene transcription abundance is likely to be directly affected by some SNP polymorphisms that alter transcription factor binding, we examined the expression of ORMDL3 and GSDMB genes in asthmatic patients and controls. Fresh peripheral blood samples from 61 patients with asthma and 70 normal subjects were collected from the Department of Respiration, Qilu Hospital, Shandong University. DNA and RNA were extracted and analyzed by real-time quantitative PCR. The expression levels of ORMDL3 and GSDMB genes were significantly higher in individuals with rs7216389, rs12603332 and rs12936231 loci than in controls (P 0.05).
Therefore, the results of this study show that: firstly, ORMDL3 gene exists as the first transcript in human peripheral blood lymphocytes, liver, esophageal cancer, colorectal cancer and lung tissues, and the different alleles of rs12603332 in the first intron of ORMDL3 gene do not affect the splicing of ORMDL3 gene mRNA. The expression levels of ORMDL3 and GSDMB genes in peripheral blood lymphocytes of adult asthmatic patients were significantly higher than those of normal controls, and the risk allele of asthma-related genetic polymorphism in 17q21 region could significantly increase the expression levels of these two genes. However, because the 17q21 region is a strong linkage disequilibrium region, screening specific functional sites from it needs more research to confirm.
The third part is the identification of the basic promoter of ORMDL3 gene.
The second part of the study showed that the abnormal expression of ORMDL3 gene may be one of the causes of asthma, and the transcriptional regulation mechanism of ORMDL3 gene is still unclear, so we analyzed the basic promoter of ORMDL3 gene in this part.
Firstly, we cloned the region from the transcription starting point of ORMDL3 gene to about 1.5 KB upstream, then inserted it into the pGL3 basic vector and analyzed its promoter activity with a dual luciferase reporter gene system. Then we used a series of truncation methods to find the smallest region containing the basic promoter of ORMDL3 gene. At 62 bp, the activity of luciferase decreased by 70% to 80%, indicating that the response element affecting the activity of ORMDL3 promoter should be located between - 68 BP and - 62 bp. There is a signal transduction and activation transcription factor 6 (STAT6) binding site between - 64 BP and - 56 bp upstream of the starting point. EMSA and super shift experiments show that STAT6 can bind to the promoter of ORMDL3 gene in vitro. ChIP experiments also show that STAT6 can bind to the promoter of ORMDL3 gene in vivo.
The results showed that the basic promoter region of ORMDL3 gene was located within - 68 bp upstream of the transcription initiation point, and a signal transduction and activation transcription factor 6 (STAT6) binding site between - 64 BP and - 56 bp upstream of the transcription initiation point played a major role in the basic promoter activity of ORMDL3 gene.
The fourth part is the regulation of ORMDL3 gene expression by STAT6.
Since STAT6 binding sites in the promoter region of ORMDL3 gene play a major role in the basic promoter activity of ORMDL3 gene, how does STAT6 regulate the expression of ORMDL3 gene? For this reason, we constructed the expression vector of STAT6 and transfected STAT6 expression vector or STAT6 siRNA in Jurkat cells, respectively. It was found that high or low expression of STAT6 could significantly activate or suppress the activity of luciferase reporter gene and the expression of endogenous ORMDL3 gene mRNA in the basic promoter of ORMDL3 gene. Since STAT6 is a transcription factor regulated by cytokine IL-4/IL-13, the expression of ORMDL3 gene is also regulated by IL-4/IL-13. Therefore, we also examined the effect of IL-4/IL-13 on ORMDL3 gene expression, and found that IL-4/IL-13 treatment significantly activated the luciferase reporter gene activity of ORMDL3 gene promoter and the expression of endogenous ORMDL3 gene mRNA. Once STAT6 was interfered with, this phenomenon would disappear, indicating that IL-4/IL-13 on ORMDL3 gene expression. In addition, some studies have found that P300 can bind to the promoter of ORMDL3 gene, and its binding site is basically the same as the STAT6 binding site we found. Therefore, we carried out ChIP experiments and found that P300 can indeed bind to the promoter of ORMDL3 gene. IP experiment
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R562.25

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