ORMDL3在哮喘發(fā)生中的作用及其轉錄調控機制研究
發(fā)布時間：2018-08-13 08:29
【摘要】：哮喘是一種由環(huán)境和遺傳因素共同作用導致的復雜疾病,其遺傳率約為36%-79%。2007年,對哮喘進行的第一個全基因組關聯(lián)分析發(fā)現(xiàn)了一個新的哮喘易感基因----ORMDL3。研究者發(fā)現(xiàn)ORMDL3所在的17q21區(qū)域中的遺傳多態(tài)位點與兒童哮喘顯著相關,并且在EB病毒感染的哮喘兒童的淋巴母細胞系中,該區(qū)域中的遺傳多態(tài)還與ORMDL3基因的表達水平密切相關。 ORMDL3基因位于17q21,是ORM基因家族的一員。在酵母中,該家族含有ORMDL1和ORMDL2兩個成員,而在人類中還有ORMDL3,共三個家族成員。在酵母中,該基因家族與神經(jīng)鞘磷脂代謝平衡相關,是神經(jīng)鞘磷脂合成過程中第一個限速酶的負調控者,提示神經(jīng)鞘磷脂代謝可能在哮喘的發(fā)生過程中具有重要作用。另外,在酵母中敲除ORM基因,表現(xiàn)為生長緩慢和對有毒物質的敏感性增強,這一現(xiàn)象在轉染入人類ORMDL3基因的mRNA后得以拯救。ORMDL3基因編碼一個含有153個氨基酸的內質網(wǎng)四次跨膜蛋白,其在人類組織中表達廣泛,特別是在肝臟和外周血淋巴細胞中高表達。研究表明ORMDL3在內質網(wǎng)上與鈣泵共定位,抑制鈣泵的功能,影響鈣離子平衡并且能夠促進內質網(wǎng)應激和未折疊蛋白反應,表明ORMDL3可能參與內質網(wǎng)有關的炎癥反應。另外,人們還發(fā)現(xiàn)在兒童臍帶血單核細胞中17q21區(qū)域中的遺傳多態(tài)不但與ORMDL3, GSDMA基因的表達水平相關,而且還與IL-17的分泌相關,表明該區(qū)域可能在免疫系統(tǒng)成熟的早期階段與T細胞的發(fā)育分化有關。而除了與哮喘相關之外,人們還發(fā)現(xiàn)ORMDL3與克隆恩病、強直性脊柱炎、系統(tǒng)性紅斑狼瘡、Ⅰ型糖尿病、神經(jīng)膠質瘤、原發(fā)性膽汁肝硬化、過敏性鼻炎等多種炎癥性疾病顯著相關。 自從發(fā)現(xiàn)ORMDL3是一個新的哮喘易感基因,在很短的時間內,人們又在多個不同種族人群中進行了驗證,結果發(fā)現(xiàn)ORMDL3基因在不同人群中均與兒童哮喘顯著相關。但是所有這些研究都是在兒童哮喘中進行的,那么,ORMDL3基因是否也與成人哮喘相關呢?在EB病毒感染的哮喘兒童淋巴母細胞系中17q21上的遺傳多態(tài)與ORMDL3基因的表達相關,那么在成人哮喘患者的外周血淋巴細胞中這一現(xiàn)象是否仍然存在呢?哮喘患者體內ORMDL3基因的表達異常是怎樣引起的呢?ORMDL3基因的表達調控又是怎樣的呢?帶著這些問題,本論文進行了以下四個方面的研究： 第一部分17q21區(qū)域與中國漢族成人哮喘的關聯(lián)分析 為了明確ORMDL3基因所在的17q21區(qū)域與成人哮喘之間的相關性,我們從山東大學齊魯醫(yī)院呼吸科收集了710例無親緣關系的成人哮喘患者,以及從健康體檢中心隨機收集了656例無親緣關系、無哮喘病及其它呼吸道疾病及過敏病史的健康體檢者作為對照組。根據(jù)相關報道及中國人群中該區(qū)域的連鎖不平衡情況,我們從17q21區(qū)域選取了5個單核苷酸多態(tài)位點(SNP):rs7216389, rs12603332, rs12936231, rs9303277, rs11557467進行基于群體的病例對照關聯(lián)分析。 結果發(fā)現(xiàn)這5個SNP多態(tài)位點均與成人哮喘顯著相關(P0.05),其中兩個SNP多態(tài)位點rs9303277, rs11557467與成人哮喘的相關性最強(P0.001)。帶有rs11557467位點G等位基因的個體或者rs9303277位點C等位基因的個體的哮喘患病風險顯著升高(OR=1.27,95%CI1.07-1.51, P=0.006以及OR=1.27,95%CI1.07-1.49,P=0.005)。即使是在去除性別和年齡的影響以后,這種相關性仍然存在。此外,單體型分析發(fā)現(xiàn),與單體型TCCCG相比,單體型CTGTT且有保護效應,OR=0.81,95%CI0.67-0.97, P=0.02。 因此,本部分研究結果表明：ORMDL3基因所在的17q21區(qū)域中的遺傳多態(tài)在中國漢族人群中與成人哮喘顯著相關。 第二部分17q21區(qū)域導致哮喘易感的功能SNP的鑒定 由于在Ensembl數(shù)據(jù)庫中顯示ORMDL3基因具有2種轉錄本形式：ENST00000304046(ORMDL3-001)以及ENST00000394169(ORMDL3-002),并且這兩個轉錄本的編碼區(qū)域是完全相同的,不同的只是非翻譯區(qū),因此它們所編碼的蛋白質是完全一樣的。那么在人體中,ORMDL3基因是以哪一種轉錄本形式存在呢?另外,Moffatt等人的研究發(fā)現(xiàn),在EB病毒感染的哮喘兒童的淋巴母細胞系中17q21區(qū)域中的遺傳多態(tài)與ORMDL3基因的表達水平密切相關,因而提示遺傳多態(tài)調節(jié)ORMDL3基因表達的異常很可能在哮喘的發(fā)生中起著一定的作用。但是到目前為止,已經(jīng)發(fā)現(xiàn)的該區(qū)域中與哮喘相關的SNP中沒有一個是位于ORMDL3基因的編碼區(qū),而我們第一部分選取的SNP位點rs12603332恰好位于該基因的第一內含子中,而已有報道提示位于內含子中的遺傳多態(tài)可能會影響特定細胞或組織中基因的轉錄剪接。因此,我們首先檢測了ORMDL3基因在人體不同組織中所使用的轉錄本形式以及該基因第一內含子中的一個SNP位點rs12603332的不同等位基因是否影響ORMDL3基因mRNA的剪接。 我們選取rs12603332位點基因型為CC及TT的個體各5例,同時選取人體肝臟組織,食管癌組織,大腸癌組織以及肺組織各12例,分別提取DNA和RNA。另外,根據(jù)兩個轉錄本的剪接特點設計引物,用PCR擴增的方法進行檢測：一對引物能同時擴增出ORMDL3的兩種轉錄本形式,用以確定ORMDL3基因是否表達同時監(jiān)控cDNA的質量,另一對引物只能擴增出第二轉錄本,用于檢測第二轉錄本是否存在。結果顯示,在人體外周血淋巴細胞、肝臟組織、食管癌組織、大腸癌組織以及肺組織中都只表達ORMDL3基因的第一轉錄本形式：ENST00000304046(ORMDL3-001),并且不論是CC基因型還是TT基因型(rs12603332位點)的個體都只表達第一轉錄本。因此,rs12603332位點并不影響ORMDL3基因的剪接,那么,該區(qū)域中的遺傳多態(tài)導致哮喘的機制是什么呢?哪個位點才是真正的功能位點呢? 由于基因轉錄水平的改變是介導哮喘或其他疾病易感的一個非常重要的機制,并且基因轉錄豐度很可能受到某些改變轉錄因子結合的SNP多態(tài)位點的直接影響。因此,我們檢測了哮喘患者和正常對照中ORMDL3基因和GSDMB基因的表達情況。從山東大學齊魯醫(yī)院呼吸科收集了61例確診的成人哮喘患者以及70例正常體檢個體的新鮮外周血,提取DNA及RNA,運用實時定量PCR的方法進行等位基因差異表達分析。結果發(fā)現(xiàn),哮喘患者外周血淋巴細胞中ORMDL3基因以及GSDMB基因的表達水平顯著高于正常對照個體,P0.05,并且攜帶有rs7216389,rs12603332以及rs12936231位點危險等位基因的個體,其ORMDL3和GSDMB基因的表達水平顯著升高,P值均小于0.05。 因此,本部分研究結果表明：首先,在人體外周血淋巴細胞、肝臟組織、食管癌組織、大腸癌組織以及肺組織中,ORMDL3基因以第一轉錄本的形式存在,并且該基因第一內含子中rs12603332位點的不同等位基因并不影響ORMDL3基因mRNA的剪接。其次,成人哮喘患者外周血淋巴細胞中ORMDL3及GSDMB基因的表達水平明顯高于正常對照,并且17q21區(qū)域中與哮喘相關的遺傳多態(tài)的危險等位基因能顯著升高這兩個基因的表達水平。因此,ORMDL3及GSDMB基因的表達異常很可能是該區(qū)域誘發(fā)哮喘的潛在機制。但由于17q21區(qū)域是一個強連鎖不平衡的區(qū)域,從中篩選出具體的功能位點尚有待于更多的研究來證實。 第三部分ORMDL3基因基本啟動子的鑒定 第二部分研究結果表明,ORMDL3基因的表達異常很可能是導致哮喘發(fā)生的原因之一,而關于該基因的轉錄調控機制目前尚不明確,因此在本部分中我們對該基因的基本啟動子進行了分析。 首先,我們克隆了ORMDL3基因轉錄起始點至上游約1.5kb的區(qū)域,連入pGL3basic載體中,用雙熒光素酶報告基因系統(tǒng)分析其啟動子活性。此后運用系列截短的辦法尋找包含ORMDL3基因基本啟動子的最小區(qū)域。結果發(fā)現(xiàn),當我們把序列從-68bp截短到-62bp時,熒光素酶活性迅速下降了70%到80%,表明影響ORMDL3基因啟動子活性的反應元件應該位于-68bp到-62bp之間。為了明確哪個轉錄因子結合在這一區(qū)域中影響ORMDL3基因的啟動子活性,我們用轉錄因子預測軟件TESS進行分析,結果發(fā)現(xiàn)在轉錄起始點上游-64bp到-56bp區(qū)域之間存在一個信號轉導及激活轉錄因子6(STAT6)的結合位點。EMSA及super shift實驗表明,在體外情況下STAT6能結合在ORMDL3基因的啟動子上。同時ChIP實驗也表明,在體內情況下STAT6也能結合在ORMDL3基因的啟動子上。 本部分實驗結果表明,ORMDL3基因的基本啟動子區(qū)域位于轉錄起始點上游-68bp以內,并且轉錄起始點上游-64bp到-56bp區(qū)域之間的一個信號轉導及激活轉錄因子6(STAT6)的結合位點對ORMDL3基因的基本啟動子活性起主要作用。 第四部分STAT6對ORMDL3基因的表達調控作用 既然ORMDL3基因啟動子區(qū)域中的STAT6結合位點對ORMDL3基因的基本啟動子活性起主要作用,那么STAT6對ORMDL3基因的表達調控作用具體是怎樣的呢?為此,我們構建了STAT6的表達載體,在Jurkat細胞中分別轉染STAT6的表達載體或者STAT6的siRNA,結果發(fā)現(xiàn)STAT6的高表達或者低表達能顯著轉錄激活或者轉錄抑制ORMDL3基因基本啟動子的熒光素酶報告基因活性及內源性ORMDL3基因mRNA的表達。由于STAT6是一個受細胞因子IL-4/IL-13調控的轉錄因子,那么ORMDL3基因的表達是否也受IL-4/IL-13的調控呢?因此,我們還檢測了IL-4/IL-13對ORMDL3基因表達的影響,結果發(fā)現(xiàn)IL-4/IL-13處理能顯著激活ORMDL3基因啟動子的熒光素酶報告基因活性及內源性ORMDL3基因mRNA的表達,而一旦干擾了STAT6,這一現(xiàn)象就會消失,表明IL-4/IL-13對ORMDL3基因的表達調控作用依賴于STAT6。另外,有研究發(fā)現(xiàn)p300也能結合在ORMDL3基因的啟動子上,并且它的結合位點與我們發(fā)現(xiàn)的STAT6結合位點基本一致。因此,我們進行了ChIP實驗,結果發(fā)現(xiàn)p300確實也能結合在ORMDL3基因的啟動子上。于是我們又進一步進行了IP實驗和ChIP-Re-ChIP實驗,結果發(fā)現(xiàn)p300和STAT6能相互結合,并且它們是以復合物的形式結合在ORMDL3基因的啟動子上。事實上,p300是一個協(xié)同轉錄因子,它并不直接結合在DNA序列上,而是通過與其他序列特異的轉錄因子結合而被招募到啟動子上。 因此,本部分研究結果表明,轉錄因子STAT6能轉錄激活ORMDL3基因的表達,并且IL-4/IL-13處理能顯著激活ORMDL3基因的啟動子活性及內源性ORMDL3基因mRNA的表達,并且這種激活作用依賴于STAT6。事實上,轉錄因子STAT6通過招募協(xié)同轉錄因子p300,共同調控ORMDL3基因的表達。這一研究為ORMDL3與炎癥及哮喘相關的事實提供了一個合理的解釋,同時也為臨床上預防和治療哮喘提供了一個新的途徑。
[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
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R562.25
本文編號：2180431
[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
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R562.25
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