Integrated analysis of miRNA and mRNA expression profiles in 2-, 6-, and 12-month-old Small Tail Han Sheep ovaries reveals that oar-miR-432 downregulates RPS6KA1 expression

Abstract

Small Tail Han Sheep are an excellent local sheep breed in China, and their outstanding reproductive performance is one of their very important biological characteristics. Clarifying the ovary development process of these ewes should provide a theoretical basis for improving their reproductive efficiency. In this study, we identified the differentially expressed (DE) microRNAs (miRNAs) in 2-, 6-, and 12-month-old small-tail Han sheep ovaries by constructing and analyzing the miRNA expression profiles. These findings clarify the molecular mechanisms regulating the excellent reproductive performance of small-tail Han ewes. We used RNA-Seq technology and bioinformatic to analyze these profiles. Eleven, 13, and 19 DE miRNAs were identified in the 2- vs 6-, 6- vs 12-, and 2- vs 12-month-old ovaries, respectively. In total, 54, 37, and 198 predicted target genes of these DE miRNAs were identified in these three groups, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that in the 2- vs 6-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 102 GO terms and seven signaling pathways; in the 6- vs 12-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 52 GO terms and three signaling pathways; and in the 2- vs 12-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 88 GO terms and six signaling pathways. Three miRNA–target regulatory networks were constructed based on these DE miRNA–target interactions. Nine miRNAs were selected to confirm to the accuracy of the miRNA sequencing data with qRT-PCR. The site at which oar-miR-432 binds RPS6KA1 was determined with a dual-luciferase system. This is the first integrated analysis the expression profiles of miRNAs and their targets during ovarian development in small-tail Han sheep. These data clarify the molecular regulatory mechanisms underlying sheep ovarian development and identify biomarkers that influence the reproductive performance of small-tail Han ewes.