Sequencing and Differential Analysis of miRNA in the Ovaries of Kazakh Horses During Estrus and Anestrus States.

Abstract: To reveal the regulatory role of miRNA in the estrous cycle transition of Kazakh horse ovaries and improve their reproductive efficiency, this study took the ovaries of Kazakh horses in the estrus period (DY group) and the quiescent period (DB group) as research objects. Small RNA sequencing and bioinformatics analysis were used to systematically analyze the expression characteristics and functional pathways of miRNA under the two physiological states. A total of 161 million high-quality miRNA tags were obtained through sequencing. Principal component analysis showed that the intra-group repeatability of the two groups of samples was good, and the expression patterns between the groups were significantly different. A total of 259 differentially expressed miRNAs (DEmiRNAs) were identified between the two groups, including eca-miR-21, eca-miR-378, eca-miR-142-3p, eca-miR-196a, and eca-miR-889, among which 99 were significantly up-regulated and 160 were significantly down-regulated. After screening with |logFC| ≥ 1.5, ≤ 0.05 and < 0.05 as strict multiple-testing correction thresholds, a total of 227 valid DEmiRNAs were retained, including 86 upregulated and 141 downregulated miRNAs; the remaining 32 transcripts showed nominal differential expression only without statistical significance after FDR correction. GO functional enrichment indicated that the differentially expressed miRNAs were mainly involved in biological processes such as cellular processes, biological regulation, molecular binding, and catalytic activity. KEGG enrichment showed that they were significantly enriched in pathways closely related to follicular development, ovulation, and luteal function, such as the Ras signaling pathway and actin cytoskeleton regulation pathway. The RT-qPCR verification results were highly consistent with the sequencing data, confirming the reliability of the analysis results. The study demonstrated that the differentially expressed miRNAs could regulate processes such as cell proliferation, apoptosis, steroidogenesis, and follicular development by targeting key signaling pathways, mediating the sequential transition of the estrous-anestrus state of Kazakh horse ovaries. The present study aimed to identify differentially expressed miRNAs in ovarian tissues of Kazakh mares between anestrus (DB) and estrus (DY) states via small RNA sequencing, reveal the miRNA-mediated molecular regulatory mechanisms of seasonal ovarian cycle transition, and provide candidate molecular markers and theoretical basis for genetic improvement of reproductive performance in Kazakh indigenous horses.