發(fā)布時間：2018-04-24 10:41

  本文選題：GWAS + 長鏈非編碼對RNA��； 參考：《南京醫(yī)科大學》2016年博士論文

【摘要】：胃癌是消化系統(tǒng)中最常見的惡性腫瘤。全球范圍內(nèi),胃癌位列全部腫瘤發(fā)病率第五位,死亡率第三位。在中國,胃癌發(fā)病率和死亡率均占消化系統(tǒng)腫瘤首位,男女發(fā)病比例約為2：1,給我國帶來嚴重的經(jīng)濟負擔和社會壓力。由于胃癌發(fā)病隱匿,且缺乏早期臨床特征,大部分患者確診時已處于進展期；加之目前尚缺乏根治性治療方案,胃癌患者易發(fā)生復發(fā)和轉移,預后生存效果不佳。因此,加強胃癌病因學和發(fā)病機制研究,篩選高效、特異、敏感的生物標志物用于胃癌的預防和治療,顯得尤為重要和迫切。大量流行病資料顯示,胃癌發(fā)生發(fā)展是多基因、多因素參與的多階段逐步演變的過程,是環(huán)境因素、生物因素以及遺傳因素、表觀遺傳因素等共同作用的結果。越來越多研究發(fā)現(xiàn),遺傳易感性研究策略為探討腫瘤病因學和發(fā)病機制提供新的思路。全基因組關聯(lián)性研究(genome-wide association study, GWAS)作為研究腫瘤易感基因的重要手段,發(fā)現(xiàn)PLCE1、MUC1和PSCA等眾多易感基因參與了胃癌發(fā)病過程,為胃癌病因學研究提供重要線索。然而,鑒于GWAS芯片“標簽位點”設計特點以及單個位點分析策略,(GWAS發(fā)現(xiàn)的易感位點僅能解釋較小部分的遺傳度,掩蓋了大量可能具有顯著胃癌發(fā)病風險指示作用的易感位點�！昂驡WAS"研究策略的提出是對GWAS的一項重要補充措施。精細作圖分析、生物信息學分析、功能學注釋以及細胞分子生物學實驗等作為“后GWAS"主要研究方案,不僅彌補了缺失的遺傳信息,而且從生物學角度闡釋了易感基因及位點參與腫瘤發(fā)病的作用機制。本研究將基于“后GWAS"研究策略,根據(jù)基因遺傳變異分布特征,采用基因集分析(gene-set analysis),深入挖掘胃癌GWAS數(shù)據(jù)；采用多階段、多種族人群病例-對照研究設計,進一步明確胃癌易感位點；結合生物信息學分析、公用數(shù)據(jù)庫分析以及一系列細胞分子生物學實驗體系,系統(tǒng)揭示胃癌發(fā)病風險的可能生物學機制。第一部分基于GWAS的非編碼RNA遺傳變異與胃癌發(fā)病風險研究目前,尚未見基于基因類型開展遺傳變異分布特征注釋的研究報道。在本研究開展前期,我們基于GENCODE基因注釋庫,對來源1000 Genomes計劃的全基因組遺傳變異位點、GWAS catalog的腫瘤易感位點以及胃癌高度相關易感位點進行分布特征注釋,發(fā)現(xiàn)雖然全基因水平上的基因間“荒漠區(qū)”位點所占比例最大,但腫瘤易感相關位點的分布逐漸向蛋白編碼與ncRNA聯(lián)合基因(簡稱：聯(lián)合基因)偏移,尤其在胃癌中,聯(lián)合基因上易感位點所占比例已高達62.1%。我們推測,聯(lián)合基因在胃癌發(fā)生發(fā)展過程中可能發(fā)揮著重要生物學調(diào)控作用�；谏鲜隹蒲屑僬f,我們對胃癌GWAS數(shù)據(jù)按照蛋白編碼基因、ncRNA基因、聯(lián)合基因以及基因間分布進行基因集分析,發(fā)現(xiàn)聯(lián)合基因上位點集合效應與胃癌易感相關性最為顯著(P=1.14×10-24)。該結果再次提示聯(lián)合基因與胃癌發(fā)生發(fā)展關系密切,為胃癌病因學研究提供思路。為進一步確定聯(lián)合基因集中的胃癌易感基因,我們在以各基因為單位的基因集分析中,發(fā)現(xiàn)一個新型的胃癌易感基因GCLET (gastric cancer low-expressed transcript)。通過功能學注釋、獨立效應分析、自然進化選擇分析以及多階段、多種族人群(亞洲人種：共5,332病例、6,452對照；歐美人種：254病例、2,835對照)的關聯(lián)性分析,發(fā)現(xiàn)具有潛在生物學效應的GCLET rs3850997 TG位點可顯著降低胃癌發(fā)病風險(OR=0.87, Pmeta=5.34×10-7)。隨后,我們通過胃癌組織學驗證以及報告基因、EMSA和ChIP等細胞分子生物學實驗,進一步探討rs3850997可能具有的等位基因特異性轉錄活性。研究發(fā)現(xiàn),G等位基因能夠降低抑制性轉錄因子CTCF的親和力,進而促進GCLET基因轉錄,實現(xiàn)對胃癌易感的保護效應。本研究是首次對胃癌GWAS開展的基因集分析,為“后GWAS"時代的研究提供重要方法借鑒和數(shù)據(jù)參考,并為易感位點的遺傳調(diào)控機制研究提供重要思路和線索。第二部分IncRNA GCLET參與胃癌發(fā)生發(fā)展的分子機制研究大量研究結果表明,長鏈非編碼RNA (long noncoding RNA, IncRNA)作為表觀遺傳學中重要組成部分,參與了細胞生長、分化等生理過程以及包括惡性腫瘤在內(nèi)的復雜疾病的病理過程。本部分對第一部分關聯(lián)性研究定位的易感基因IncRNA GCLET開展深入的分子作用機制研究以探討其在胃癌發(fā)生發(fā)展過程的重要調(diào)控作用。我們結合臨床流行病學資料,分析GCLET與臨床表型相關性；并觀察GCLET對細胞惡性表型的影響。此外,通過報告基因、細胞干擾和Western blot等細胞分子生物學實驗,以及胃癌組織學驗證,檢測胞質(zhì)中高豐度IncRNA GCLET是否具有“海綿”樣作用吸附miRNAs,發(fā)揮“競爭內(nèi)源性RNA(competing endogenous RNA, ceRNA)"效應。研究發(fā)現(xiàn),IncRNA GCLET對胃癌細胞惡性增殖、侵襲和遷移具有顯著抑制效應,進而參與了胃癌病程進展(浸潤深度Ptrend=0.037;淋巴轉移Ptrend=0.019;臨床分期Ptrend=0.003)以及預后轉歸(logrank p-0.005)。生物信息學分析和細胞分子生物學實驗證實,GCLET可競爭性結合miR-27a-3p,抑制其對下游靶基因FOXP2的翻譯活性,形成"GCLET-miR-27a-3p-FOXP2"作用通路參與胃癌發(fā)生發(fā)展。本部分研究所開展的臨床表型與細胞表型結果相輔相成,互為驗證,為GCLET作為臨床治療效果評價、病程跟蹤和預后判斷的潛在生物標志物提供重要參考;同時深入細致的分子作用機制研究也為GCLET臨床應用價值的實現(xiàn)提供理論支撐。
[Abstract]:Gastric cancer is the most common malignant tumor in the digestive system. In the world, the total incidence of cancer is fifth and the mortality rate is third. In China, the incidence and mortality of gastric cancer account for the first of the digestive system tumors, and the incidence of male and female is about 2:1, which brings serious economic burden and social pressure to our country. In addition to the lack of early clinical features, most of the patients have been in progress at the time of diagnosis. In addition, there is still a lack of radical treatment, and the patients with gastric cancer are prone to recurrence and metastasis, and the survival effect is poor. Therefore, the study of the etiology and pathogenesis of gastric cancer and the screening of high effective, specific and sensitive biomarkers for the prevention of gastric cancer are screened. A large number of epidemiological data show that the development of gastric cancer is a multistage and gradual process of multi gene and multi factor participation, which is the result of the common effects of environmental factors, biological factors, genetic factors and epigenetic factors. More and more studies have found that the strategy of genetic susceptibility research is a discussion. The etiology and pathogenesis of cancer provide new ideas. Genome-wide association study (GWAS) is an important means for the study of tumor susceptibility genes. It is found that many susceptible genes such as PLCE1, MUC1 and PSCA are involved in the pathogenesis of gastric cancer and provide important clues for the study of gastric cancer. However, GWAS chips have been found. "Label loci" design features and single locus analysis strategies (GWAS found that the susceptible loci can only explain the heritability of smaller parts and cover a large number of susceptible loci that may have significant indications of the risk of gastric cancer. "Post GWAS" research strategy is an important supplement to GWAS. Informatics analysis, functional annotation and cell molecular biology experiment as the "post GWAS" main research scheme not only make up for the missing genetic information, but also explain the mechanism of the susceptibility genes and loci involved in the tumor pathogenesis from a biological point of view. This study will be based on the "post GWAS" strategy, based on the genetic variation of genes. Gene-set analysis was used to explore the GWAS data of gastric cancer, and a multi stage, multi population case control study was designed to further clarify the susceptibility loci of gastric cancer. Combined with bioinformatics analysis, public database analysis and a series of cell molecular biological experiments, the incidence of gastric cancer was systematically revealed. Possible biological mechanisms of risk. Part one is based on the study of GWAS based non coded RNA genetic variation and the risk of gastric cancer. There has not been a report on genetic variation distribution characteristics annotation based on genetic types. In the early stage of this study, we based on the GENCODE gene annotation library for the whole genome of the source 1000 Genomes program. It was found that the susceptibility loci of GWAS catalog and the highly sensitive loci of gastric cancer were annotated. It was found that although the proportion of the "desert area" loci was the largest in the whole gene level, the distribution of the tumor susceptibility loci gradually shifted to the protein encoding and the ncRNA gene (abbreviated as the joint gene). Especially in gastric cancer, the proportion of the susceptible loci in the joint gene is up to 62.1%.. We speculate that the combined gene may play an important biological control role in the development of gastric cancer. Based on the above hypothesis, the GWAS data of gastric cancer are based on the protein encoding gene, ncRNA gene, joint gene and INTERGENE distribution. The gene set analysis showed that the association between the combined gene and the susceptibility to gastric cancer was most significant (P=1.14 * 10-24). The results suggest that the association gene is closely related to the development of gastric cancer and provides ideas for the study of the etiology of gastric cancer. In the gene set analysis, a new gastric cancer susceptibility gene GCLET (gastric cancer low-expressed transcript) was found. Through functional annotation, independent effect analysis, natural evolutionary selection analysis and multistage, multiple ethnic groups (Asian races: 5332 cases, 6452 controls; European and American races: 254 cases, 2835 controls) GCLET rs3850997 TG loci with potential biological effects were found to significantly reduce the risk of gastric cancer (OR=0.87, Pmeta=5.34 10-7). Then, we further explored the possible allelic specific transcriptional activity of rs3850997, through the histological examination of gastric cancer and the molecular biological experiments of the reported genes, EMSA and ChIP. The study found that the G allele can reduce the affinity of the inhibitory transcription factor CTCF, and then promote the GCLET gene transcription and realize the protective effect on the susceptibility to gastric cancer. This study is the first gene set analysis for the gastric cancer of the gastric cancer, which provides important methods for reference and data reference for the research of "post GWAS" time and the inheritance of susceptible loci. The study of regulatory mechanism provides important ideas and clues. Second the molecular mechanisms of IncRNA GCLET involved in the development of gastric cancer have shown that long chain non coded RNA (long noncoding RNA, IncRNA), as an important part of epigenetics, participates in cell growth, differentiation and other physiological processes, as well as malignant tumors. The pathological process of complex diseases, including IncRNA GCLET, the first part of the correlation study, is carried out to explore the important regulatory mechanism of the molecular mechanism in the development of gastric cancer. We combine the clinical epidemiological data to analyze the correlation between GCLET and clinical phenotype, and observe the GCLET In addition, the effects of "competitive endogenous RNA (competing endogenous RNA, ceRNA)" on the effect of "competitive endogenous RNA (competing endogenous RNA, ceRNA)" were detected by the detection of the high abundance IncRNA GCLET in the cytoplasm with the "sponge" like effect of miRNAs by the reporter gene, cell interference and Western blot cell molecular biology experiments, as well as the histological examination of gastric cancer. It is found that IncRNA GCLET has a significant inhibitory effect on the malignant proliferation, invasion and migration of gastric cancer cells, and then participates in the progression of gastric cancer (infiltration depth Ptrend=0.037, lymphatic metastasis Ptrend=0.019, clinical staging Ptrend=0.003) and prognosis (logrank p-0.005). Bioinformatics analysis and cell molecular biology experiments confirmed that GCLET can be used. The competitive combination of miR-27a-3p, which inhibits the translation activity of the target gene FOXP2 to the downstream target gene, forms a "GCLET-miR-27a-3p-FOXP2" pathway to participate in the development of gastric cancer. The clinical phenotype and the cell phenotype results are complementary to each other, and are mutually validated for the evaluation of the effect of the clinical treatment, the course tracking and the prognosis of the prognosis. It provides an important reference for biomarkers, and a detailed and detailed molecular mechanism study provides theoretical support for the realization of clinical application value of GCLET.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.2

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