基于GWAS的非编码RNA参与胃癌发生发展的机制研究

发布时间：2018-04-24 10:41

本文选题：GWAS + 长链非编码对RNA　；参考：《南京医科大学》2016年博士论文

【摘要】：胃癌是消化系统中最常见的恶性肿瘤。全球范围内,胃癌位列全部肿瘤发病率第五位,死亡率第三位。在中国,胃癌发病率和死亡率均占消化系统肿瘤首位,男女发病比例约为2：1,给我国带来严重的经济负担和社会压力。由于胃癌发病隐匿,且缺乏早期临床特征,大部分患者确诊时已处于进展期；加之目前尚缺乏根治性治疗方案,胃癌患者易发生复发和转移,预后生存效果不佳。因此,加强胃癌病因学和发病机制研究,筛选高效、特异、敏感的生物标志物用于胃癌的预防和治疗,显得尤为重要和迫切。大量流行病资料显示,胃癌发生发展是多基因、多因素参与的多阶段逐步演变的过程,是环境因素、生物因素以及遗传因素、表观遗传因素等共同作用的结果。越来越多研究发现,遗传易感性研究策略为探讨肿瘤病因学和发病机制提供新的思路。全基因组关联性研究(genome-wide association study, GWAS)作为研究肿瘤易感基因的重要手段,发现PLCE1、MUC1和PSCA等众多易感基因参与了胃癌发病过程,为胃癌病因学研究提供重要线索。然而,鉴于GWAS芯片“标签位点”设计特点以及单个位点分析策略,(GWAS发现的易感位点仅能解释较小部分的遗传度,掩盖了大量可能具有显著胃癌发病风险指示作用的易感位点。“后GWAS"研究策略的提出是对GWAS的一项重要补充措施。精细作图分析、生物信息学分析、功能学注释以及细胞分子生物学实验等作为“后GWAS"主要研究方案,不仅弥补了缺失的遗传信息,而且从生物学角度阐释了易感基因及位点参与肿瘤发病的作用机制。本研究将基于“后GWAS"研究策略,根据基因遗传变异分布特征,采用基因集分析(gene-set analysis),深入挖掘胃癌GWAS数据；采用多阶段、多种族人群病例-对照研究设计,进一步明确胃癌易感位点；结合生物信息学分析、公用数据库分析以及一系列细胞分子生物学实验体系,系统揭示胃癌发病风险的可能生物学机制。第一部分基于GWAS的非编码RNA遗传变异与胃癌发病风险研究目前,尚未见基于基因类型开展遗传变异分布特征注释的研究报道。在本研究开展前期,我们基于GENCODE基因注释库,对来源1000 Genomes计划的全基因组遗传变异位点、GWAS catalog的肿瘤易感位点以及胃癌高度相关易感位点进行分布特征注释,发现虽然全基因水平上的基因间“荒漠区”位点所占比例最大,但肿瘤易感相关位点的分布逐渐向蛋白编码与ncRNA联合基因(简称：联合基因)偏移,尤其在胃癌中,联合基因上易感位点所占比例已高达62.1%。我们推测,联合基因在胃癌发生发展过程中可能发挥着重要生物学调控作用。基于上述科研假说,我们对胃癌GWAS数据按照蛋白编码基因、ncRNA基因、联合基因以及基因间分布进行基因集分析,发现联合基因上位点集合效应与胃癌易感相关性最为显著(P=1.14×10-24)。该结果再次提示联合基因与胃癌发生发展关系密切,为胃癌病因学研究提供思路。为进一步确定联合基因集中的胃癌易感基因,我们在以各基因为单位的基因集分析中,发现一个新型的胃癌易感基因GCLET (gastric cancer low-expressed transcript)。通过功能学注释、独立效应分析、自然进化选择分析以及多阶段、多种族人群(亚洲人种：共5,332病例、6,452对照；欧美人种：254病例、2,835对照)的关联性分析,发现具有潜在生物学效应的GCLET rs3850997 TG位点可显著降低胃癌发病风险(OR=0.87, Pmeta=5.34×10-7)。随后,我们通过胃癌组织学验证以及报告基因、EMSA和ChIP等细胞分子生物学实验,进一步探讨rs3850997可能具有的等位基因特异性转录活性。研究发现,G等位基因能够降低抑制性转录因子CTCF的亲和力,进而促进GCLET基因转录,实现对胃癌易感的保护效应。本研究是首次对胃癌GWAS开展的基因集分析,为“后GWAS"时代的研究提供重要方法借鉴和数据参考,并为易感位点的遗传调控机制研究提供重要思路和线索。第二部分IncRNA GCLET参与胃癌发生发展的分子机制研究大量研究结果表明,长链非编码RNA (long noncoding RNA, IncRNA)作为表观遗传学中重要组成部分,参与了细胞生长、分化等生理过程以及包括恶性肿瘤在内的复杂疾病的病理过程。本部分对第一部分关联性研究定位的易感基因IncRNA GCLET开展深入的分子作用机制研究以探讨其在胃癌发生发展过程的重要调控作用。我们结合临床流行病学资料,分析GCLET与临床表型相关性；并观察GCLET对细胞恶性表型的影响。此外,通过报告基因、细胞干扰和Western blot等细胞分子生物学实验,以及胃癌组织学验证,检测胞质中高丰度IncRNA GCLET是否具有“海绵”样作用吸附miRNAs,发挥“竞争内源性RNA(competing endogenous RNA, ceRNA)"效应。研究发现,IncRNA GCLET对胃癌细胞恶性增殖、侵袭和迁移具有显著抑制效应,进而参与了胃癌病程进展(浸润深度Ptrend=0.037;淋巴转移Ptrend=0.019;临床分期Ptrend=0.003)以及预后转归(logrank p-0.005)。生物信息学分析和细胞分子生物学实验证实,GCLET可竞争性结合miR-27a-3p,抑制其对下游靶基因FOXP2的翻译活性,形成"GCLET-miR-27a-3p-FOXP2"作用通路参与胃癌发生发展。本部分研究所开展的临床表型与细胞表型结果相辅相成,互为验证,为GCLET作为临床治疗效果评价、病程跟踪和预后判断的潜在生物标志物提供重要参考;同时深入细致的分子作用机制研究也为GCLET临床应用价值的实现提供理论支撑。
[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.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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