MiRNAs‐containing extracellular vesicles (EVs) possess the unique function of mediating intercellular communication and participating in many biological processes such as post‐transcriptional gene regulation of embryo implantation and placental development. In the present study, Illumina small‐RNA sequencing was used to identify differentially expressed (DE) miRNAs in serum EVs of pregnant (P) and non‐pregnant (NP) Kazakh sheep at Day 17 from mating. The specifically and differentially expressed miRNAs at early pregnancy in sheep were verified by using RT‐PCR. The target genes of DE miRNAs were predicted by bioinformatics software, and the functional and pathway enrichment analysis was performed on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) terms. A total of 562 miRNAs (210 novel miRNAs) were identified by sequencing, of which 57 miRNAs were differentially expressed, 49 were up‐regulated, 8 were down‐regulated and 22 novel miRNAs were specifically expressed in the pregnant sheep. Eight highly expressed known miRNA (miR‐378‐3p, miR‐320‐3p, miR‐22‐3p, let‐7b, miR‐423‐3p, miR‐221, miR‐296‐3p, miR‐147‐3p) in pregnant group were down‐regulated in the control group. miRNAs‐containing pregnancy‐related terms and regulatory pathways regulation were enriched using both GO and KEGG analyses. Moreover, we also envisioned a miRNA‐mRNA interaction network to understand the function of miRNAs involved in the early pregnancy serum regulatory network. The results of RT‐PCR verification confirmed the reliability of small‐RNA sequencing. Among them, miR‐22‐3p and miR‐378‐3p were significantly differentially expressed (DE) between pregnant sheep and non‐pregnant group (p < 0.01). The site at which oar‐miR‐22‐3p binds MAPK3 was determined with a dual‐luciferase system. This is the first integrated analysis of the expression profiles of EV‐miRNAs and their targets during early pregnancy in ewes. These data identify key miRNAs that influence the implantation of sheep in the early stage of pregnancy, and provide theoretical basis for further molecular regulatory mechanisms research.

中文翻译：

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早孕期哈萨克羊血清细胞外囊泡（EVs）中差异表达miRNAs的鉴定

含有miRNAs的细胞外囊泡（EVs）具有独特的介导细胞间通讯和参与许多生物学过程的功能，如胚胎着床和胎盘发育的转录后基因调控。在本研究中，Illumina 小 RNA 测序用于鉴定在交配后第 17 天怀孕（P）和未怀孕（NP）哈萨克绵羊血清 EV 中的差异表达（DE）miRNA。通过RT-PCR验证了绵羊妊娠早期特异性和差异表达的miRNA。通过生物信息学软件预测DE miRNA的靶基因，利用基因本体论（GO）和京都基因与基因组百科全书（KEGG）术语进行功能和通路富集分析。通过测序共鉴定出 562 个 miRNA（210 个新的 miRNA），其中57个miRNA差异表达，49个上调，8个下调，22个新的miRNA在妊娠绵羊中特异性表达。8 个高表达的已知 miRNA（miR-378-3p、miR-320-3p、miR-22-3p、let-7b、miR-423-3p、miR-221、miR-296-3p、miR-147-3p）妊娠组在对照组中下调。使用 GO 和 KEGG 分析丰富了含有 miRNA 的妊娠相关术语和调控途径调控。此外，我们还设想了一个 miRNA-mRNA 相互作用网络，以了解参与早期妊娠血清调节网络的 miRNA 的功能。RT-PCR验证结果证实了小RNA测序的可靠性。其中，miR-22-3p和miR-378-3p在孕羊与未孕组之间存在显着差异表达（DE）。p  < 0.01)。oar-miR-22-3p 结合 MAPK3 的位点是用双荧光素酶系统确定的。这是对母羊早期妊娠期间 EV-miRNA 及其靶标表达谱的首次综合分析。这些数据确定了影响羊妊娠早期着床的关键miRNA，为进一步的分子调控机制研究提供了理论依据。