·长篇论著·

基于生物信息学探讨肥胖与不孕症的关系

李丹丹 王东梅 曹卫平 翟凤婷

【摘要】 目的 通过生物信息学技术方法探讨肥胖与不孕症的关系。 方法 在GeneCards、TTD、OMIM疾病数据库中挖掘并筛选肥胖和不孕症的相关基因,结合Uniprot数据库得到相关基因靶点,去重、映射后得到关键靶点,基于STRING数据库构建关键靶点的PPI网络图,筛选出核心靶点,利用DAVID数据库进行GO和KEGG富集分析。 结果 搜索与筛选不孕症相关靶点312个及肥胖相关靶点483个,包括关键靶点61个,以Degree值≥16筛选出核心靶点17个,得到33项GO富集结果及13条KEGG信号通路。 结论 胰岛素、促阿片-黑素细胞皮质素原、瘦素、白细胞介素6、肿瘤坏死因子等核心靶点通过脂肪细胞因子信号通路、Janu激酶/信号转导与转录激活子信号通路、胰岛素抵抗信号通路、腺苷酸激活蛋白激酶信号通路、卵巢类固醇生成信号通路、磷脂酰肌醇3-激酶/蛋白激酶B信号通路、哺乳动物雷帕霉素靶蛋白信号通路等多个通路对两个疾病起到调控作用。肥胖与不孕症两疾病之间内分泌通讯是双向的,本文以期为后续机制进一步的探讨和实验设计的优化等提供思路。

【关键词】 不孕症; 肥胖; 生物信息学; 相互关系

不孕症(infertility)是一种由多种病因导致的生育障碍状态,女性无避孕性生活至少12个月而未孕称为不孕症[1],其发病趋势逐年上升。世界卫生组织预测,21世纪不孕症将成为仅次于肿瘤和心脑血管病的第三大疾病[2]。肥胖作为常见的慢性代谢性疾病之一,“正像流行性疾病一样蔓延、吞噬整个世界”。肥胖(obesity)是体内脂肪过度蓄积和(或)分布异常,往往伴有体重增加[3]。近年来,中国肥胖率也迅速增长。有研究发现,肥胖与多囊卵巢综合征(polycystic ovary syndrome,PCOS)存在某种影响,可降低妊娠率,增高流产率,最终改变妊娠结局[4]

中医古籍中也有肥胖与不孕的相关记载。金元·朱震亨《丹溪心法》言:“若是肥盛妇人,禀受甚厚,恣于酒食之人,经水不调,不能成胎,谓之躯脂满溢,闭塞子宫”。明·万全《万氏妇人科》云:“惟彼肥硕者,膏脂充满,玄室之户不开,挟痰者痰涎壅滞,血海之波不流……及为浊、为带、为经闭、为无子之病”。清·傅山《傅青主女科》载:“妇人有身体肥胖,痰涎甚多,不能受孕者……湿盛者多肥胖,肥胖者多气虚,气虚者多痰涎……日积月累,则胞胎竟变为汪洋之水窟矣。且肥胖之妇,内肉必满,遮隔子宫,不能受精,此必然之势也”。

因而肥胖与不孕症间有着密切的关系,本文利用生物信息技术从微观角度探讨两疾病间的关系,有助于更好地理解疾病发生、发展的相关机制,同时也为临床进一步探究提供了思路。

资料与方法

一、肥胖和不孕症的疾病靶点筛选与核心靶点预测

在GeneCards、TTD、OMIM数据库中搜索关键词:“infertility”、“obesity”,结合Uniprot数据库得到疾病相关靶点,去重、映射得到关键靶点,通过STRING数据库构建关键靶点的PPI网络图,将图用Cytoscape3.7.2进行分析,根据节点度(Degree)值≥16筛选核心作用靶点。

二、生物通路及富集分析

利用DAVID数据库对核心靶点进行Gene Ontology(GO)和 Kyoto Encyclopedia of Genes and Genomes(KEGG)富集分析,筛选出与研究疾病相关的前10个GO条目与KEGG条目,依据Omicshare云平台制作高级气泡图,通过富集结果分析两种疾病间的关系。

结 果

经过搜索、标准化处理、查重整合后共选出不孕症相关靶点312个,肥胖相关靶点483个,包括关键靶点61个,见韦恩图(见图1)。构建出关键靶点的PPI网络图(见图2),靶点之间有384条相互作用关系,Degree值范围1到32,筛选到核心靶点17个(见图3),核心靶点即是研究不孕症与肥胖联系的重要节点。利用DAVID数据库进行分析,P值代表富集的显著性,P<0.05代表富集结果显著[5],FDR为矫正后P值(<0.05),共筛选出33项GO条目及13条KEGG通路,以P值升序排列,分别筛选出符合条件的10个GO、KEGG条目,并制作气泡图(见图4、图5)。生物过程及通路下所含靶点分别见表1、表2。

图1 不孕症与肥胖的映射关键靶点韦恩图

Figure 1 Venn diagram of key targets for mapping infertility and obesity

图2 关键靶点的PPI互相作用网络图

Figure 2 PPI interaction network diagram of key targets

图3 核心靶点网络图(核心靶点大小为Degree值大小)

Figure 3 Core target network diagram (the size of the core target is the size of the Degree value)

图4 GO富集气泡图

Figure 4 The bubble diagram of GO enrichment analysis

图5 KEGG富集气泡图

Figure 5 The bubble diagram of KEGG enrichment pathways

表1 GO富集分析(与疾病相关的前10条)

Table 1 GO enrichment analysis (top 10 disease-related)

Biological process Target genePositive regulation of MAPK cascadeAR, IL6, LEP, IGFBP3, IGF2, IGF1, INSHormone activityPOMC, LEP, IGF2, GNRH1, PRL, IGF1, INSGlucose homeostasisPOMC, IL6, LEP, LEPR, PTPN11, INSExtracellular regionPOMC, IL6, LEP, IGFBP3, LEPR, IGF2, GNRH1, PRL, IGF1, TNF, INSOsitive regulation of peptidyl-tyrosine phosphorylationIL6, IGF2, IGF1, TP53, INSPositive regulation of protein kinase B signalingIL6, LEP, IGF2, TNF, INSInsulin receptor bindingIGF2, PTPN11, IGF1, INSCellular protein metabolic processIGFBP3, IGF2, PRL, IGF1, INSExtracellular spacePOMC, IL6, LEP, IGFBP3, IGF2, GNRH1, IGF1,TNF, INSNegative regulation of lipid storageIL6, LEP, TNF

表2 KEGG通路(与疾病相关的前10条)

Table 2 KEGG enrichment pathways (top 10 disease-related)

Biological process Target geneAdipocytokine signaling pathwayPOMC, LEP, LEPR, PTPN11, TNFJak-STAT signaling pathwayIL6, LEP, LEPR, PTPN11, PRLNon-alcoholic fatty liver disease (NAFLD)IL6, LEP, LEPR, TNF, INSProstate cancerAR, IGF1, TP53, INSInsulin resistanceIL6, PTPN11, TNF, INSCytokine-cytokine receptor interactionIL6, LEP, LEPR, PRL, TNFAMPK signaling pathwayLEP, LEPR, IGF1, INSOvarian steroidogenesisIGF1, CYP19A1, INSPI3K-Akt signaling pathwayIL6, PRL, IGF1, TP53, INSmTOR signaling pathwayIGF1, TNF, INS

讨 论

近年来,肥胖对生育力的影响逐渐受到关注。在青少年和年轻女性中,肥胖的发病年龄和月经异常的发病年龄显著相关[6]。有研究表明,体重指数(body mass index,BMI)是评价育龄妇女生育能力的一个重要指标[7],接受体外受精(in vitro fertilization,IVF)/卵胞浆内单精子注射(intracytoplasmic sperm injection,ICSI)的高BMI组患者早期流产率显著高于正常体重组,提示过度肥胖对IVF/ICSI结局有一定的负面影响[8]。因而肥胖与月经周期不规律、生育能力降低、流产风险增加之间存在显著关联。本文应用生物信息学技术在挖掘两疾病的相关基因并对其分析,得到同时作用于两疾病的核心靶点,胰岛素(insulina,INS)、促阿片-黑素细胞皮质素原(pro-opiomelanocortin,POMC)、胰岛素样生长因子1(insulin like growth factor 1,IGF1)、瘦素(leptin,LEP)、白细胞介素6(interleukin 6,IL6)、肿瘤坏死因子(tumor necrosis factor,TNF)等。GO分析从生物学过程、细胞学组分、分子生物学功能三个功能大类,对一个基因的功能进行多方面的限定和描述,发现两疾病主要涉及丝裂原活化蛋白激酶(MAPK)级联的正调控、激素的活性、葡萄糖稳态、胰岛素受体结合、脂质储存的负调控等。分析KEGG通路结果,发现不孕与肥胖的核心靶点主要通过作用于脂肪细胞因子信号通路、Janu激酶/信号转导与转录激活子(JAK/STAT)信号通路、胰岛素抵抗、腺苷酸激活蛋白激酶(AMP-activated Protein Kinase,AMPK)信号通路、卵巢类固醇生成、磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路、哺乳动物雷帕霉素靶蛋白(mTOR)信号通路。

LEP和脂联素(adiponectin,APN)是脂肪细胞分泌的两大重要的细胞因子[9]。其中LEP可以调节代谢、内分泌功能、免疫功能等,可以抑制食欲,促进能量消耗,影响糖代谢。LEP与瘦素受体(leptin receptor,LEPR)通过偶联和激活酪氨酸激酶(Janus kinases,JAK)2经多种信号转导途径作用于POMC。其信号转导通路有JAK/STAT信号转导通路、MAPK/细胞外信号调节激酶1/2(ERK1/2)信号转导通路、胰岛素受体底物(IRS)/PI3K信号转导通路3种途径[10-13]。JAK-STAT信号通路与PI3K/AKT/mTOR信号通路协同发挥作用[14]。炎症因子刺激可激活JAK-STAT,进而影响巨噬细胞的分化和炎症因子的表达[15]。该信号通路紊乱,则会引起一些相关的免疫疾病,如甲状腺疾病、糖尿病[16]。LEP能减弱肝细胞系中酪氨酸胰岛素诱导的信号,拮抗部分INS功能,从而产生胰岛素抵抗(insulin resistance,IR)[17]。IR是很多代谢性疾病的一个重要特征,糖尿病所导致的高雄激素血症及IR在PCOS的发病中起着早期和决定性的作用[18]。中国PCOS患者合并肥胖的患病率为34.1%~43.3%[19]。肥胖和IR会破坏窦卵泡发育,从而导致慢性无排卵[20],腹部肥胖影响尤其显著,其机制包括INS介导的卵巢类固醇生成过度刺激和性激素结合球蛋白血浓度降低[21]。肥胖人群有高LEP,通过干扰促性腺激素的刺激作用,抑制黄体化的颗粒细胞产生雌激素和孕激素[22]。另外,LEP可调节促性腺激素释放激素(gonadotropin-releasing hormone, GnRH)的分泌,慢性过度肥胖会产生瘦素抵抗,减少GnRH分泌,降低生育能力[23]。APN可以增强INS作用,影响糖脂代谢,也有抗动脉粥样硬化、炎性反应等作用[24],其水平在肥胖人群中显著降低,与肥胖症、脂质代谢异常、糖尿病、心血管疾病明显相关[25]。有研究证明,缺乏APN会导致减弱其介导的INS信号转导,从而出现IR[26]。APN通过激活AMPK信号通路可抑制GnRH和黄体生成素(luteinizing hormone,LH)的释放,调节下丘脑-垂体-卵巢(HPO)轴,又导致子宫内膜容受性降低,影响生殖代谢功能[27-29]。AMPK信号通路是调节细胞能量状态的中心环节,AMPK通过LEP、ANP等因子在增加葡萄糖摄取、增强INS敏感性等方面发挥重要作用。多种脂源性细胞因子表达异常与IR密切相关,继而影响2型糖尿病(diabetes mellitus type 2,T2DM)的发生发展[30]

脂肪组织还能分泌炎症细胞因子,如肿瘤坏死因子(TNF-α)、IL-6等[30]。TNF-α可通过抑制INS信号转导、抑制葡萄糖的转运和摄取、降低INS受体数目及其与INS的亲合力、加速脂肪分解等导致全身性IR[25]。IL-6也可通过介导免疫炎性反应参与其中。肥胖人群体内长期存在代谢性炎症[31],此状态不仅影响了卵母细胞正常的生长、成熟,导致卵母细胞质量下降,引起胚胎发育不良[32]。还会通过加重子宫内膜炎症影响子宫内膜蜕膜化而降低其容受性,增加流产率[33]。在免疫反应中,细胞因子如TNF-α等可诱导巨噬细胞表达,调控其功能[34]。巨噬细胞在调控母体免疫状况的过程中发挥着重要作用,通过调节母体免疫耐受来影响胚胎能否免受免疫打击而流产[35-38]。细胞自噬过程是溶酶体对自身结构的吞噬降解,mTOR是调节细胞生长和增殖的重要因子,是自噬启动阶段的关键调节因子,可抑制自噬发生,主要靶点mTOR相关靶点包括TOR复合体1和Ⅰ型PI3K,因此巨噬细胞自噬的调控,其可能的信号通路为PI3K/Akt/mTOR通路[35,39-40]

多个研究也表明LEP等多个脂源性细胞因子通过非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)信号通路引起代谢综合征(metabolic syndrome,MS),与IR关系密切。

本文依托生物信息技术,探究肥胖与不孕症两疾病之间的关系,两者间发生发展与机体内分泌改变关系密切,脂肪组织和生殖系统之间的内分泌通讯是双向的,功能性脂肪组织能够调节生殖健康[41]。肥胖可在不同水平影响卵母细胞成熟、胚胎发育和着床、胎盘和子宫环境[42]。此类基因靶点的信息整合依赖于生物信息技术、基因数据库的准确性,具体作用机制仍需进一步试验研究的验证。

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To explore the relationship between obesity and infertility based on bioinformatics

LI Dandan, WANG Dongmei, CAO Weiping, ZHAI Fengting. The First Clinical Medical College of Shandong University of Traditional Chinese Medicine; Department of Gynecology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250000, China

[Abstract] Objective To explore the relationship between obesity and infertility through bioinformatics techniques. Methods The genes related to infertility and obesity were mined and screened in GeneCards, TTD and OMIM disease databases. The related gene targets were obtained by combining Uniprot database, and the key targets were obtained by deduplicating and mapping. PPI network diagrams of the key targets were constructed based on STRING database, and the core targets were selected for GO and KEGG enrichment analysis with DAVID database. Results There were 312 infertility related targets and 483 obesity-related targets, including 61 key targets. 17 core targets were screened with Degree value ≥16, 33 GO enrichment results, and 13 KEGG signaling pathways were obtained. Conclusion INS, POMC, LEP, IL6, TNF and other core targets play a regulatory role in the two diseases through adipocytokine, JAK-STAT, insulin resistance, AMPK, ovarian steroid generation, PI3K-Akt, mTOR and other signaling pathways. Endocrine communication between obesity and infertility is bidirectional. We aims to provide ideas for further discussion of the follow-up mechanism and optimization of experimental design.

[Key words] infertility; obesity; bioinformatics; interrelationship

【中图分类号】 R17; R71

基金项目:山东省自然科学基金(ZR2019MH083);王东梅山东省名老中医药专家传承工作室

作者单位:250000 济南,山东中医药大学第一临床医学院(李丹丹);山东中医药大学附属医院妇科(王东梅,曹卫平,翟凤婷)

通信作者:王东梅(ds_yx2020@163.com)

(收稿日期:2020-11-23)