摘要
目的探讨胎儿羊水外泌体中miRNA在唐氏综合征(DS)胎儿生长发育中的作用。方法收集DS胎儿羊水和正常胎儿羊水,分别提取羊水外泌体miRNA。设置对照组:正常胎儿羊水外泌体miRNA;DS组:DS胎儿羊水外泌体miRNA。运用miRNA测序技术筛选出两组中差异表达的miRNA,并对差异表达的miRNA进行靶基因预测及功能分析(GO)和信号通路分析。从差异表达的miRNA中挑选3个与DS表型最相关的miRNA进行qPCR验证。通过双荧光素酶报告基因技术验证let-7d-5p对BACH1的靶向调控作用。结果和对照组相比,DS组中存在15个差异表达的miRNA,其中表达上调的miRNA有7个,表达下调的miRNA有8个。靶基因预测结果发现差异表达的miRNA可以靶向调控17种与DS相关的基因。GO分析发现靶基因主要功能与蛋白结合、蛋白转运、ATP结合、转移酶活性、突触等有关。Pathway通路分析发现富集显著的功能通路与神经系统发育有着密切的联系。qPCR验证结果发现与对照组相比,DS组中miR-140-3p、let-7d-5p水平显著降低(P<0.05),与测序结果一致;而DS组中miR-4512水平较对照组显著增加(P<0.05),与测序结果相反。双荧光素酶报告基因检测结果证实let-7d-5p可靶向调控BACH1的表达。结论羊水外泌体let-7d-5p可能通过调控BACH1的表达促进DS胎儿大脑氧化应激事件。
Objective To investigate the role of miRNAs in amniotic fluid exosomes in growth and development of fetuses with Down syndrome(DS).Methods Amniotic fluid were collected from 20 fetuses with DS and 20 normal fetuses(control)to extract amniotic exosome miRNA.MicroRNA sequencing technique was used to identify the differentially expressed miRNAs between the two groups,for which gene ontology(GO)and pathway analysis was performed.Three differentially expressed miRNAs with the strongest correlation with DS phenotype were selected for qPCR verification.Dual luciferase reporter assay was used to verify the activity of let-7d-5p for targeted regulation of BACH1.Results We identified 15 differentially expressed miRNAs in DS as compared with the control group,among which 7 miRNAs were up-regulated and 8 were down-regulated.Target gene prediction results showed that the differentially expressed miRNAs targeted 17 DS-related genes.GO analysis revealed that the main functions of the target genes involved protein binding,protein transport,ATP binding,transferase activity and synapses.Pathway analysis revealed that the functional pathways were closely related with the development of the nervous system.qPCR results showed that the expression levels of miR-140-3p and let-7d-5p were significantly lower in DS group than in the control group(P<0.05),as was consistent with miRNA sequencing results;the expression level of miR-4512 was significantly higher in DS group than in control group(P<0.05),which was contrary to miRNA sequencing results.The results of double luciferase reporter gene assay confirmed that let-7d-5p was capable of targeted regulation of BACH1 expression.Conclusion Let-7d-5p in amniotic fluid exosomes may promote oxidative stress events in the brain of fetuses with DS by regulating BACH1 expression.
作者
丁凯泽
余蕾
黄智
郑慧玲
杨雪
田甜
谢汝佳
DING Kaize;YU Lei;HUANG Zhi;ZHENG Huiling;YANG Xue;TIAN Tian;XIE Rujia(Department of Pathophysiology,Guizhou Medical University,Guiyang 550025,China;Department of Assisted Reproduction,Guiyang Maternal and Child Health Care Center,Guiyang 550003,China;Department of Pathology,Guiyang Maternal and Child Health Care Center,Guiyang 550003,China;Department of Eugenic Genetics,Guiyang Maternal and Child Health Care Center,Guiyang 550003,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2022年第2期293-299,共7页
Journal of Southern Medical University
基金
贵州省科技计划项目(黔科合支撑[2020]4Y126号)
贵阳市高层次创新型青年卫生人才培养计划项目(【2020】筑卫健科技合同字第016号)。
