鸡胚外泌体miRNAs功能分析

doi:10.11843/j.issn.0366-6964.2020.010

摘要/Abstract

摘要： 旨在基于鸡胚外泌体及所含有的miRNAs解析其产生生理功效的活性物质基础及作用机制。本研究收集13胚龄惠阳胡须鸡胚蛋18枚（3组，每组6枚），采用酶消化、蔗糖密度梯度和差速超速离心等方法分离鸡胚组织来源的外泌体，经透射电镜、纳米流式和标志蛋白流式检测等方法鉴定其特征。利用高通量测序方法研究外泌体样本中miRNAs表达谱，对各样本top 10且共表达的miRNAs进行靶基因预测与功能分析。结果表明，分别获得了3个浮力密度范围（S1：1.13 g·mL^-1、S2：1.21 g·mL^-1和S3：1.32 g·mL^-1）具有外泌体典型特征的胞外囊泡；与miRBase V21进行比对发现，S1、S2和S3样品中分别含有675、661和845个已知的miRNAs，这些miRNAs中有488个（52.6%）三者共有，包括了广泛参与细胞增殖、分化和免疫应答等过程的功能性miRNAs。TargetScan 7.2和miRDB预测得到3个样本中top 10且共表达的56个miRNAs的靶基因，获得的3 650个靶基因经DAVID分析，显著富集了24个与机体生长、发育、免疫等有关的生物学通路（P<0.05），包括MAPK信号通路、mTOR信号通路、Notch信号通路、Wnt信号通路和GnRH信号通路等。本研究建立了鸡胚组织中有效分离外泌体的方法，发现鸡胚组织来源的外泌体miRNAs会影响机体生长发育和免疫调节有关的生物学通路。

关键词: 鸡胚, 外泌体, 分离, 功能研究

Abstract: This study was conducted to elucidate the physiological basis of active substances and their mechanism of chicken embryos based on exosomes and their miRNAs. Eighteen Huiyang Beard chicken embryos with 13 embryo age were selected (3 groups, 6 eggs per group), and embryos-derived exosomes were isolated by enzymatic digestion, sucrose cushion gradient and differential ultracentrifugation, and their characteristics were identified by transmission electron microscopy, nanoflow and labeled protein flow detection. High-throughput sequencing was used to study the expression profile of miRNAs in exosome samples, and target genes prediction and functional analysis were performed for top 10 and co-expressed miRNAs. The results showed that extracellular vesicles with typical characteristics of exosomes were obtained from 3 buoyant density ranges (S1:1.13 g·mL^-1, S2:1.21 g·mL^-1 and S3:1.32 g·mL^-1), respectively. A total of 675, 661 and 845 known chicken miRNAs of S1, S2 and S3 samples were detected via alignment to miRBase V21, in which 488 (52.6%) miRNAs were co-expressed in the 3 samples. These co-expressed miRNAs might participate in the cell proliferation, differentiation, and immune response, etc. Based on both TargetScan 7.2 and miRDB systems, a total of 3 650 genes could be targeted the 56 miRNAs which were top 10 and co-expressed in the 3 samples. In addition, the 3 650 target genes were analyzed by DAVID, it was found that 24 pathways related to growth, development and immunity were significantly enriched by these genes (P<0.05), which including MAPK signaling pathway, mTOR signaling pathway, Notch signaling pathway, Wnt signaling pathway and GnRH signaling pathway, etc. In this study, an effective method to isolate exosomes from chicken embryo tissue was established, and chicken embryos-derived exosome miRNAs could affect biological pathways related to growth, development and immune regulation.

Key words: chicken embryos, exosome, isolation, functional study

中图分类号:

S831.3

李莹, 王艳, 王劼, 何静怡, 罗成龙, 瞿浩. 鸡胚外泌体miRNAs功能分析[J]. 畜牧兽医学报, 2020, 51(6): 1260-1270.

LI Ying, WANG Yan, WANG Jie, HE Jingyi, LUO Chenglong, QU Hao. Functional Analysis of microRNAs in Chicken Embryos-derived Exosomes[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1260-1270.

参考文献 50

[1]	李曦.鸡胚蛋对免疫力低下小鼠的免疫调节功能研究[D].无锡:江南大学,2012.LI X.Study on the immunomodulatroy effect of chick embryo egg on hypoimmunity mice[D].Wuxi:Jiangnan University,2012.(in Chinese)
[2]	刘娟娟.不同品种鸡胚蛋孵化期间营养物质的变化[D].泰安:山东农业大学,2007.LIU J J.Changes of the nutrients in different types of embryonated eggs during incubation peroid[D].Tai'an:Shandong Agricultural University,2007.(in Chinese)
[3]	段翔.孵化对鸡蛋营养成分及生物活性组分的影响[D].无锡:江南大学,2015.DUAN X.Effect of fertilization on nutritional compositions and bioactive components of hen egg[D].Wuxi:Jiangnan University,2015.(in Chinese)
[4]	李利东,袁建兴,宓晓黎,等.不同孵化期的鸡胚蛋营养和功效成分研究[J].食品科学,2004,25(11):287-290.LI L D,YUAN J X,MI X L,et al.Study on nutrients improvement and efficacy factors of embrynated eggs duing different incubation periods[J].Food Science,2004,25(11):287-290.(in Chinese)
[5]	任军甫.鸡胚孵化过程中营养成分变化规律的研究与胶囊研制[D].洛阳:河南科技大学,2011.REN J F.Study on the changes of nutrition during incubation of chick embryo eggs and development of capsule[D].Luoyang:Henan University of Science and Technology,2011.(in Chinese)
[6]	DUAN X,LI M,WU F F,et al.Postfertilization changes in nutritional composition and protein conformation of hen egg[J].J Agric Food Chem,2013,61(49):12092-12100.
[7]	张丽静,付劢,陈继兰,等.北京油鸡胚蛋孵化过程中营养成分变化[J].食品工业科技,2019,40(19):291-295, 300.ZHANG L J,FU M,CHEN J L,et al.Changes of nutritional ingredients in embryonated Beijing fatty chicken eggs during its incubating[J].Science and Technology of Food Industry,2019,40(19):291-295, 300.(in Chinese)
[8]	王倩,王化洲,齐志敏,等.鸡胚口服液免疫调节作用的实验研究[J].中国生化药物杂志,2000,21(4):195-197.WANG Q,WANG H Z,QI Z M,et al.An experimental study on the effect of the per oral solution extracted from chicken embryo on immunologic function[J].Chinese Journal of Biochemical Pharmaceutics,2000,21(4):195-197.(in Chinese)
[9]	吕玉琴,郝志明,李引乾,等.鸡胚对小鼠生长和免疫力的影响[J].西安联合大学学报,2002,5(2):26-29.LÜ Y Q,HAO Z M,LI Y Q,et al.Chick embryo impact on mouse growth and immunity[J].Journal of Xi'an United University, 2002, 5(2):26-29.(in Chinese)
[10]	郝志明,谢斐,吕玉琴.鸡胚复方制剂对小白鼠生长、脾脏指数及血浆SOD和MDA的影响[J].西安联合大学学报, 2004, 7(2):8-11.HAO Z M,XIE F,LÜ Y Q,et al.Chicken embryo compound preparation on mouse's growth,spleen index,SOD & MDA plasma[J]. Journal of Xi'an United University,2004,7(2):8-11.(in Chinese)
[11]	陈正跃,黄丽君,许建文,等.鸡胚胎素的美容抗衰老及免疫调节作用(英文)[J].中国临床康复,2006,10(27):190-192.CHEN Z Y,HUANG L J,XU J W,et al.Effect of eggembryosin on cosmetology,anti-senility and immunoloregulation[J].Chinese Journal of Clinical Rehabilitation,2006,10(27):190-192.(in Chinese)
[12]	李丹.鸡胚及其人参配伍免疫调节与抗疲劳作用的研究[D].泰安:山东农业大学,2008.LI D.Study on immune regulatory and anti-fatigue effect of chicken embryo and combined with Ginseng[D].Tai'an:Shandong Agricultural University,2008.(in Chinese)
[13]	VLASSOV A V,MAGDALENO S,SETTERQUIST R,et al.Exosomes:current knowledge of their composition,biological functions, and diagnostic and therapeutic potentials[J].Biochim Biophys Acta,2012,1820(7):940-948.
[14]	LOBB R J,BECKER M,WEN S W,et al.Optimized exosome isolation protocol for cell culture supernatant and human plasma[J]. J Extracell Vesicles,2015,4:27031.
[15]	倪爱心,麻慧,陈继兰.寄生虫来源的外泌体研究进展[J].畜牧兽医学报,2019,50(5):909-917.NI A X,MA H,CHEN J L.Research progress of parasite-derived exosomes[J].Acta Veterinaria et Zootechnica Sinica,2019, 50(5):909-917.(in Chinese)
[16]	GALLEY J D,BESNER G E.The therapeutic potential of breast milk-derived extracellular vesicles[J].Nutrients, 2020, 12(3):E745.
[17]	THÉRY C,ZITVOGEL L,AMIGORENA S.Exosomes:composition,biogenesis and function[J].Nat Rev Immunol,2002, 2(8):569-579.
[18]	CESSELLI D,PARISSE P,ALEKSOVA A,et al.Extracellular vesicles:how drug and pathology interfere with their biogenesis and function[J].Front Physiol,2018,9:1394.
[19]	LÄSSER C,ELDH M,LÖTVALL J.Isolation and characterization of RNA-containing exosomes[J].J Vis Exp,2012(59):e3037.
[20]	LÖNNERDAL B.Human milk MicroRNAs/exosomes:composition and biological effects[J].Nestle Nutr Inst Workshop Ser,2019,90:83-92.
[21]	CORRADO C,RAIMONDO S,CHIESI A,et al.Exosomes as intercellular signaling organelles involved in health and disease:basic science and clinical applications[J].Int J Mol Sci,2013,14(3):5338-5366.
[22]	JURJ A,ZANOAGA O,BRAICU C,et al.A comprehensive picture of extracellular vesicles and their contents.Molecular transfer to cancer cells[J].Cancers (Basel),2020,12(2):E298.
[23]	GU Y R,LI M Z,WANG T,et al.Lactation-related microRNA expression profiles of porcine breast milk exosomes[J].PLoS One,2012,7(8):e43691.
[24]	WOLF T,BAIER S R,ZEMPLENI J.The intestinal transport of bovine milk exosomes is mediated by endocytosis in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells[J].J Nutr,2015,145(10):2201-2206.
[25]	IZUMI H,TSUDA M,SATO Y,et al.Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages[J].J Dairy Sci,2015,98(5):2920-2933.
[26]	张顺华.猪不同泌乳期乳汁Exosome中microRNA转录组的鉴定和表达谱分析[D].雅安:四川农业大学,2013.ZHANG S H.Lactation-related microRNA expression profiles of porcine breast milk exosomes[D].Ya'an:Sichuan Agricultural University,2013.(in Chinese)
[27]	ZHANG L,CHEN T,YIN Y L,et al.Dietary microRNA-A novel functional component of food[J].Adv Nutr,2019,10(4):711-721.
[28]	ZENG J,GUPTA V K,JIANG Y M,et al.Cross-kingdom small RNAs among animals,plants and microbes[J].Cells,2019,8(4):371.
[29]	MA J D,WANG C D,LONG K R,et al.Exosomal microRNAs in giant panda (Ailuropoda melanoleuca) breast milk:potential maternal regulators for the development of newborn cubs[J].Sci Rep,2017,7(1):3507.
[30]	TENG Y,REN Y,SAYED M,et al.Plant-derived exosomal MicroRNAs shape the gut microbiota[J].Cell Host Microbe, 2018, 24(5):637-652.e8.
[31]	BAIER S R,NGUYEN C,XIE F,et al.MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells,HEK-293 kidney cell cultures,and mouse livers[J].J Nutr,2014,144(10):1495-1500.
[32]	ZHOU Z,LI X H,LIU J X,et al.Honeysuckle-encoded atypical microRNA2911 directly targets influenza A viruses[J].Cell Res, 2015,25(1):39-49.
[33]	PEREZ-GONZALEZ R,GAUTHIER S A,KUMAR A,et al.The exosome secretory pathway transports amyloid precursor protein carboxyl-terminal fragments from the cell into the brain extracellular space[J].J Biol Chem,2012,287(51):43108-43115.
[34]	VELLA L J,SCICLUNA B J,CHENG L,et al.A rigorous method to enrich for exosomes from brain tissue[J].J Extracell Vesicles,2017,6(1):1348885.
[35]	LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods,2001,25(4):402-408.
[36]	GALLART-PALAU X,SERRA A,SZE S K.Enrichment of extracellular vesicles from tissues of the central nervous system by PROSPR[J].Mol Neurodegener,2016,11(1):41.
[37]	ZHANG Y,YU M,DAI M,et al.miR-450a-5p within rat adipose tissue exosome-like vesicles promotes adipogenic differentiation by targeting WISP2[J].J Cell Sci,2017,130(6):1158-1168.
[38]	PÉREZ-GONZÁLEZ R,GAUTHIER S A,KUMAR A,et al.A method for isolation of extracellular vesicles and characterization of exosomes from brain extracellular space[M]//HILL A.Exosomes and Microvesicles.Methods in Molecular Biology,vol 1545.New York:Humana Press,2017:139-151.
[39]	GAO F,JIAO F,ZHANG Y,et al.Advances in separation techniques for exosomes and their clinical applications[J].Se Pu,2019,37(10):1071-1083.
[40]	LIU Z G,CAUVI D M,BERNARDINO E M A,et al.Isolation and characterization of human urine extracellular vesicles[J].Cell Stress Chaperones,2018,23(5):943-953.
[41]	DONG P,ZHANG Y,YAN D Y,et al.Protective effects of human milk-derived exosomes on intestinal stem cells damaged by oxidative stress[J].Cell Transplant,2020,29:1-8.
[42]	DOYLE L M,WANG M Z.Overview of extracellular vesicles,their origin,composition,purpose,and methods for exosome isolation and analysis[J].Cells,2019,8(7):727.
[43]	林德麟,陈婷,黎梦,等.乳中miRNA的研究进展[J].畜牧兽医学报,2016,47(9):1739-1748.LIN D L,CHEN T,LI M,et al.Research progress of miRNA in milk[J].Acta Veterinaria et Zootechnica Sinica,2016, 47(9):1739-1748.(in Chinese)
[44]	SUN Y,LIU W Z,LIU T,et al.Signaling pathway of MAPK/ERK in cell proliferation,differentiation,migration, senescence and apoptosis[J].J Recept Signal Transduct Res,2015,35(6):600-604.
[45]	张秀秀,郭云涛,黄万龙,等.靶向MAPK信号通路调控脂肪细胞分化的microRNAs[J].畜牧兽医学报,2016,47(11):2159-2166.ZHANG X X,GUO Y T,HUANG W L,et al.Regulation of adipocyte differentiation via microRNAs targeting MAPK signaling pathway[J].Acta Veterinaria et Zootechnica Sinica,2016,47(11):2159-2166.(in Chinese)
[46]	郑琪,睢梦华,凌英会.骨骼肌卫星细胞增殖与成肌分化过程中关键信号通路的作用[J].畜牧兽医学报,2017, 48(11):2005-2014.ZHENG Q,SUI M H,LING Y H.The role of key signaling pathways in the proliferation and differentiation of skeletal muscle satellite cells[J].Acta Veterinaria et Zootechnica Sinica,2017,48(11):2005-2014.(in Chinese)
[47]	HWANG Y S,SEO M,LEE B R,et al.The transcriptome of early chicken embryos reveals signaling pathways governing rapid asymmetric cellularization and lineage segregation[J].Development,2018,145(6):dev157453.
[48]	LUO W,LIN S M,LI G H,et al.Integrative Analyses of miRNA-mRNA interactions reveal let-7b,miR-128 and MAPK pathway involvement in muscle mass loss in sex-linked dwarf chickens[J].Int J Mol Sci,2016,17(3):276.
[49]	NUTHALAPATI N K,EVANS J D,TAYLOR R L,et al.Transcriptomic analysis of early B-cell development in the chicken embryo[J].Poult Sci,2019,98(11):5342-5354.
[50]	WEICHHART T,HENGSTSCHLÄGER M,LINKE M.Regulation of innate immune cell function by mTOR[J].Nat Rev Immunol,2015,15(10):599-614.