IRS-2、CDKN1A基因微小RNA靶序列结合区单核苷酸多态性与多囊卵巢综合征的相关性研究

发布时间：2018-02-26 16:31

本文关键词： 胰岛素受体底物2 周期蛋白依赖激酶抑制因子1A 多囊卵巢综合征微小RNA 单核苷酸多态性　出处：《暨南大学》2016年硕士论文　论文类型：学位论文

【摘要】：多囊卵巢综合征(PCOS)是一种常见的以月经紊乱、持续性排卵障碍和卵巢多囊样改变为主要临床特征,常合并高雄激素血症(HA)和胰岛素抵抗(IR)及代偿性的高胰岛素血症(HI)的生殖功能障碍与代谢异常并存的内分泌代谢紊乱综合症。PCOS属于生殖系统和内分泌代谢性疾病,越来越多的研究表明PCOS是一种累及全身、危害女性终生健康的基因与环境共同作用的遗传相关性疾病。目前有关PCOS病因和发病机理的研究有诸多报道,但其病因和确切发病机制仍不十分清楚。PCOS发病常常表现为家族聚集性,诸多学者认为遗传因素在PCOS病因学方面起重要作用;但近年PCOS相关遗传候选基因研究仍无突破性进展。目前普遍认为PCOS是遗传基因与环境因素相互作用的结果,近年的研究关注点在表观遗传学方面领域,研究的方法集中在DNA甲基化方面。随着分子生物技术的发展,尤其是生物信息学的广泛应用,研究的关注点开始转向非编码微小RNA(miRNA)与PCOS发生发展中的关系,已有研究表明miRNA在PCOS发生过程中参与调控作用。miRNA是一种微小片段非编码RNA,主要通过与靶信使RNA(mRNA)的3’UTR区域特异性结合,在靶目标mRNA的降解、翻译抑制和脱腺苷化等转录后水平调控细胞基因的转录及表达,进而参与了生命或疾病的发生过程。尽管其不参与基因的编码,但是通过与靶RNA结合来降低其稳定性或下调它们的转录,进而参与到细胞生长、分化发育、凋亡等生物学过程中。而miRNA与其靶基因结合区域单核苷酸多态性(SNPs)与多种疾病易感性密切相关。胰岛素抵抗是PCOS的基本病理生理特征之一,研究认为IRS-2基因多态在胰岛素抵抗中扮演了重要作用,PCOS患者卵泡液中miR-135a表达上调,而潜在的miRNA目标基因IRS-2的表达下调。PCOS发生的另一个重要机制是卵巢局部微环境的改变,研究发现,miR-93以及其靶基因CDKN1A共同调节PCOS卵巢颗粒细胞的生理功能通过调节卵巢局部细胞因子,参与多囊卵巢的形成。目前,关于miRNA下游靶基因胰岛素受体底物2基因(IRS2)和周期蛋白依赖激酶抑制因子1A基因(CDKN1A/P21)结合区域SNPs与PCOS病变在人群中的关联研究报道暂时未见。基于传统的分子遗传学原理,结合表观遗传学的研究手段,本研究根据现有的相关基因的研究成果,从生物医学文献数据库筛选,采用生物信息学方法分析和预测PCOS相关的mi RNA靶结合区域内的单核苷酸多态性位点,运用先进的高通量基因测序技术进行测序,比较分析所研究对象的单核苷酸多态性位点、基因型等差异,探讨IRS2、CDKN1A基因3’UTR区域特异性结合单核苷酸多态性与PCOS相关病变的高度异质性、家族聚集现象之间的相关性,为进一步阐明pcos的发病机制、预测其发生风险,以及为pcos的临床早期防治、诊断和病情评估及预后判断提供新的参考依据。本研究采用病例-对照研究方法,通过临床上收集pcos患者及对照组的临床信息资料及外周血样本,描述临床上的基本特征及生活习惯,分析临床特征与基因多态性、基因型的关系。研究目的1.通过收集pcos患者和对照组临床基本信息以及辅助检查的数据资料,描述和分析pcos在人群当中的临床基本特征。2.通过筛选irs2、cdkn1a基因的mirnas靶结合区域内的单核苷酸多态性位点,探讨遗传多态性位点、基因型与pcos病变的相关性,从分子水平发现pcos的易感因素及潜在的危险因素。3.利用生物信息学方法,从表观遗传学角度分析基因-基因以及基因-环境之间的联合作用,阐明基因-基因以及基因-环境之间的相互作用,探讨单核苷酸多态性与pcos临床特征、异质性和家族聚集现象的关系。研究方法1.采用病例对照研究设计,选取2014年08月—2015年10月在广东省计划生育专科医院门诊就诊及住院的共188例pcos患者及对照组为研究对象,部分收集5mledta抗凝血,抽提全基因组dna,进行snps基因型检测。2.运用问卷调查方法收集调查对象基本信息及相关暴露因素。填写调查问卷,对其一般特征及临床资料进行回顾性分层对照分析,采用elisa法检测生殖相关激素,并计算卵泡刺激素/黄体生成素(fsh/lh)比值;测定空腹胰岛素(fins)及空腹血糖(fpg)值,并计算homa-ir指数;测定抗苗勒管激素;并予以阴道b超等辅助检查。3.运用spss13.0软件比较分析mir-135a、mir-93靶基因irs-2、cdkn1a结合区单核苷酸多态性与pcos及其临床特征相关性。研究结果1.临床资料回顾性分析1)pcos患者的bmi、基础lh、基础t、amh、homa-ir显著高与对照组(p0.05)。2)高雄激素血症组fins水平显著高于非高雄激素血症组(p0.05);hi组bmi、prl、fins显著高于非hi组(p0.05);肥胖组homa-ir水平显著高于非肥胖组,差异有统计学意义(p0.05);月经稀发组amh、homa-ir显著高于月经正常组,差异有统计学意义(p0.05)。2.共筛选了miRNA相关的2个靶基因7个单核苷酸多态性位点,其中IRS2基因两个位点:rs2289046和rs1865434;CDKN1A基因5个位点:rs1059234、rs3176366、rs3176337、rs3176359和rs74801436。对照组中的基因型rs2289046、rs1865434、rs1059234、rs3176337分布符合哈温平衡。另外3个单核苷酸多态性位点在本样本群体中不是多态的。3.IRS2基因2个单核苷酸多态性位点与CDKN1A基因2个单核苷酸多态性位点的三种基因型和两种等位基因频率分布在PCOS组和对照组以及PCOS组内的分层比较中的差异无统计学意义(P0.05)。研究结论1.PCOS患者临床特征多样化,符合PCOS临床诊断标准,表现异质性;胰岛素抵抗的发生与月经稀发和肥胖有关。2.暂未发现筛选的IRS2基因、CDKN1A基因miRNA靶结合区域的单核苷酸多态性位点与PCOS病变的遗传易感性具有显著相关性;调整年龄、BMI、FINS等因素后,两组之间仍未见显著相关性。
[Abstract]:Polycystic ovary syndrome (PCOS) is a kind of common to menstrual disorders, persistent anovulation and polycystic ovaries as the main clinical features, often associated with Kaohsiung hormones (HA) and insulin resistance (IR) and compensatory hyperinsulinemia (HI).PCOS metabolic disorder syndrome coexisting abnormal reproductive function the metabolic disorder and belongs to the reproductive system and the endocrine and metabolic disease, more and more studies show that PCOS is a systemic disease, genetic correlation between the harm to women's lifelong health gene environment interaction. The present research on PCOS etiology and pathogenesis of the disease there are many reports, but its etiology and pathogenesis is still not very clear the incidence of.PCOS is often a family gathering, many scholars believe that genetic factors play an important role in the etiology of PCOS; but in recent years, genetic research on genes related to PCOS is still no candidate Breakthrough. PCOS is considered to be the interaction of genetic and environmental factors, in recent years the research focus in the field of epigenetics, research methods focus on DNA methylation. With the development of molecular biological technology, especially the widespread application of bioinformatics to non encoding micro RNA began to study focus (miRNA) and PCOS in the development of the relationship, studies have shown that miRNA in the process of PCOS in.MiRNA regulation is a tiny fragment encoding RNA, mainly through the target messenger RNA (mRNA) 3 'UTR region specific binding in the degradation of target mRNA, transcription and expression translation inhibition and deadenylation and post transcriptional regulation of cell gene, and then participate in the process of life or disease. Although it is not involved in the gene encoding, but by binding to RNA to reduce its stability The qualitative or downregulation of their transcription, and then participate in cell growth, differentiation, apoptosis and other biological processes. MiRNA and its target gene with single nucleotide polymorphisms (SNPs) associated with various disease susceptibility. Insulin resistance is the basic pathophysiological features of PCOS, studies suggest that IRS-2 gene plays an important role in insulin resistance in patients with PCOS, upregulate the expression of miR-135a in follicular fluid and the expression of miRNA target gene IRS-2 potential down another important mechanism of.PCOS is the ovarian microenvironment, the study found that the physiological function of miR-93 and its target gene CDKN1A regulated PCOS ovarian granulosa cells by regulating ovarian cytokine, involved in the formation of polycystic ovary. At present, about miRNA downstream target genes of insulin receptor substrate 2 gene (IRS2) and cyclin dependent kinase inhibitor Gene 1A (CDKN1A/P21) SNPs and PCOS combined with the regional association of disease in the population reported no temporary. Based on the traditional principle of molecular genetics, combined with research methods of genetics, based on the research results of the existing related genes, screened from the biomedical literature database, using bioinformatics method to analyze and forecast PCOS the MI RNA target binding sites of single nucleotide polymorphisms within the region, were sequenced using high-throughput gene sequencing technology, comparative analysis of single nucleotide polymorphism of the object of study, genotype differences of IRS2, CDKN1A gene 3 'UTR region specific with high heterogeneity and single nucleotide polymorphism of PCOS related diseases. The correlation between the phenomenon of familial aggregation, to further elucidate the pathogenesis of PCOS, predict the risk of early prevention and treatment of PCOS and clinical diagnosis. Provide new reference for judging the evaluation and prognosis of fault and disease. In this study, a case-control study, PCOS patients and control subjects were collected by clinical data and clinical information of peripheral blood samples, describes the basic characteristics and habits and clinical analysis of clinical features and gene polymorphism, genotype. Objective: 1. patients with PCOS and control group by collecting clinical data of basic information and auxiliary examination, description and analysis of the clinical characteristics of.2. PCOS in the crowd through the screening of IRS2, miRNAs target gene CDKN1A binding sites of single nucleotide polymorphisms within the region, to explore the genetic polymorphism, relationship between genotype and PCOS lesions. From the molecular level to find the susceptible factors of PCOS and the potential risk factors of.3. using bioinformatics methods, from the perspective of epigenetics analysis of gene gene and gene environment Combined effects, elucidate the interactions between gene gene and gene environment, to investigate single nucleotide polymorphism and PCOS clinical features, the relationship between the phenomenon of heterogeneity and familial aggregation. Methods 1. case-control study from 2014 08 months to October 2015 in Guangzhou Family Planning Specialty Hospital outpatient clinic and hospitalized a total of 188 patients with PCOS and the control group as the research object, part of the collection of 5mledta anticoagulant, extraction of genomic DNA and SNPs genotypes of.2. using the questionnaire survey method to collect the basic information survey and related exposure factors. Fill in the questionnaire, the general characteristics and clinical data were retrospectively stratified control analysis, ELISA method was used to detect reproductive hormones, and the calculation of FSH / LH ratio (fsh/lh); fasting insulin (fins) and fasting blood glucose (FPG), and calculate the HOMA-IR index; Determination of anti Mullerian hormone; and vaginal examination using.3. B Ultrasound SPSS13.0 software analysis and comparison of mir-135a, mir-93 and IRS-2 target gene, CDKN1A binding between single nucleotide polymorphism of PCOS and its clinical features. Results of the 1. clinical data were retrospectively analyzed in 1 patients with PCOS) BMI, LH, t AMH, HOMA-IR, and the control group was significantly higher (P0.05).2) Kaohsiung hormones group fins level was significantly higher than that of non Kaohsiung hormones group (P0.05); group hi BMI, PRL, fins were significantly higher than that of HI group (P0.05); the level of HOMA-IR in obese group was significantly higher than that in the non obese group, the difference was statistically significant (P0.05) oligomenorrhea; group AMH, HOMA-IR group was significantly higher than that of normal menstruation, the difference was statistically significant (P0.05.2.) were screened miRNA 2 related target gene 7 SNPs, including two SNPs of IRS2 gene: rs2289046 and rs1865434; CDKN1A gene 5 sites: rs1059234, rs3176366, rs3176337, rs3176359 and rs74801436. genotype rs2289046, control group rs1865434, rs1059234, rs3176337 distribution is in accordance with Hardy Weinberg equilibrium. Three genotypes of 2 SNPs of CDKN1A gene and 3 other SNPs are not polymorphic in this sample of.3.IRS2 gene 2 nucleotide polymorphic loci and two had no statistical significance in the PCOS group and the control group and the difference between the layers in PCOS group in the gene frequency distribution (P0.05). Conclusions: 1.PCOS patients clinical characteristics of diversification, conforms to the standard of clinical diagnosis of PCOS, showing the heterogeneity; the occurrence of insulin resistance and oligomenorrhea and obesity about.2. not found IRS2 gene screening, CDKN1A gene with genetic susceptibility to miRNA target region single nucleotide polymorphism and PCOS disease have significant correlation There was no significant correlation between the two groups after the adjustment of age, BMI, FINS and other factors.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R711.75

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