Identification of differentially expressed miRNAs in serum extracellular vesicles (EVs) of Kazakh sheep at early pregnancy

Abstract 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.