State Key Laboratory for Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Science, Xinjiang, China.
College of Animal Science and Technology, Shihezi University, Shihezi, China.
Reprod Domest Anim. 2021 May;56(5):713-724. doi: 10.1111/rda.13910. Epub 2021 Mar 9.
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.
miRNAs 包含的细胞外囊泡（EVs）具有介导细胞间通讯的独特功能，并参与许多生物学过程，如胚胎着床和胎盘发育的转录后基因调控。本研究采用 Illumina 小 RNA 测序技术，鉴定了配种后第 17 天怀孕（P）和非怀孕（NP）哈萨克绵羊血清 EV 中的差异表达（DE）miRNAs。通过 RT-PCR 验证了绵羊早期妊娠中特异性和差异表达的 miRNAs。利用生物信息学软件预测 DE miRNAs 的靶基因，并对基因本体论（GO）和京都基因与基因组百科全书（KEGG）术语进行功能和途径富集分析。通过测序共鉴定了 562 个 miRNAs（210 个新 miRNAs），其中 57 个 miRNAs 差异表达，49 个上调，8 个下调，22 个新 miRNAs 在怀孕绵羊中特异性表达。在怀孕组中，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）在对照组中下调。通过 GO 和 KEGG 分析，富集了与妊娠相关术语和调控途径调节相关的 miRNAs。此外，我们还构建了一个 miRNA-mRNA 相互作用网络，以了解参与早期妊娠血清调控网络的 miRNAs 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 在怀孕绵羊和非怀孕组之间差异表达（DE）显著（p<0.01）。用双荧光素酶系统确定了 oar-miR-22-3p 与 MAPK3 结合的位点。这是首次对绵羊早期妊娠 EV-miRNAs 及其靶基因的表达谱进行综合分析。这些数据确定了影响绵羊早期妊娠着床的关键 miRNAs，为进一步研究分子调控机制提供了理论依据。
