{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T22:54:40Z","timestamp":1788821680725,"version":"build-2803163510"},"reference-count":37,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T00:00:00Z","timestamp":1784073600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Central Guided Local Science and Technology Development Fund Project","award":["ZYYD 2025 JD 02"],"award-info":[{"award-number":["ZYYD 2025 JD 02"]}]},{"name":"2025 Xinjiang Uyghur Autonomous Region \u201cSky Lake Talent\u201d Young Doctor Introduction Program"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>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 |log2FC| \u2265 1.5, p \u2264 0.05 and q &lt; 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\u2013anestrus 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.<\/jats:p>","DOI":"10.3390\/ani16142197","type":"journal-article","created":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T01:05:07Z","timestamp":1784163907000},"page":"2197","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Sequencing and Differential Analysis of miRNA in the Ovaries of Kazakh Horses During Estrus and Anestrus States"],"prefix":"10.3390","volume":"16","author":[{"given":"Jiahao","family":"Liu","sequence":"first","affiliation":[{"name":"College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinkui","family":"Yao","sequence":"additional","affiliation":[{"name":"College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China"},{"name":"Xinjiang Key Laboratory of Equine Breeding and Exercise Physiology, Urumqi 830052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Meng","sequence":"additional","affiliation":[{"name":"College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China"},{"name":"Xinjiang Key Laboratory of Equine Breeding and Exercise Physiology, Urumqi 830052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7148-4557","authenticated-orcid":false,"given":"Jianwen","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China"},{"name":"Xinjiang Key Laboratory of Equine Breeding and Exercise Physiology, Urumqi 830052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-1881-1221","authenticated-orcid":false,"given":"Yaqi","family":"Zeng","sequence":"additional","affiliation":[{"name":"College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China"},{"name":"Xinjiang Key Laboratory of Equine Breeding and Exercise Physiology, Urumqi 830052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-5822-8366","authenticated-orcid":false,"given":"Wanlu","family":"Ren","sequence":"additional","affiliation":[{"name":"College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China"},{"name":"Xinjiang Key Laboratory of Equine Breeding and Exercise Physiology, Urumqi 830052, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"eadm7506","DOI":"10.1126\/sciadv.adm7506","article-title":"Cellular atlas of the human ovary using morphologically guided spatial transcriptomics and single-cell sequencing","volume":"10","author":"Jones","year":"2024","journal-title":"Sci. Adv."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"gaae015","DOI":"10.1093\/molehr\/gaae015","article-title":"Cellular senescence of granulosa cells in the pathogenesis of polycystic ovary syndrome","volume":"30","author":"Tanaka","year":"2024","journal-title":"Mol. Hum. Reprod."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e81088","DOI":"10.7554\/eLife.81088","article-title":"Integration of mouse ovary morphogenesis with developmental dynamics of the oviduct, ovarian ligaments, and rete ovarii","volume":"11","author":"McKey","year":"2022","journal-title":"eLife"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Liu, J., Yang, Y., Wen, L., Wen, M., Zeng, Y., Ren, W., and Yao, X. (2026). Transcriptome Sequencing and Differential Analysis of Testes of 1-Year-Old and 3-Year-Old Kazakh Horses. Biology, 15.","DOI":"10.3390\/biology15010100"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Wubulikasimu, M., Liu, J., Yao, X., Meng, J., Wang, J., Zeng, Y., Li, L., and Ren, W. (2025). Transcriptomic sequencing and differential analysis of Kazakh horse muscles from various anatomical locations. Front. Vet. Sci., 12.","DOI":"10.3389\/fvets.2025.1633786"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"146228","DOI":"10.1016\/j.ijbiomac.2025.146228","article-title":"Differential analysis of testicular LncRNA in Kazakh horses of different ages","volume":"321","author":"Lu","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Ren, W., Wang, J., Zeng, Y., Wang, T., Meng, J., and Yao, X. (2024). Differential age-related transcriptomic analysis of ovarian granulosa cells in Kazakh horses. Front. Endocrinol., 15.","DOI":"10.3389\/fendo.2024.1346260"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1016\/j.semcancer.2021.05.004","article-title":"miRNA dysregulation is an emerging modulator of genomic instability","volume":"Volume 76","author":"Cardoso","year":"2021","journal-title":"Seminars in Cancer Biology"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.semcancer.2020.05.017","article-title":"Dissecting miRNA facilitated physiology and function in human breast cancer for therapeutic intervention","volume":"Volume 72","author":"Sengupta","year":"2021","journal-title":"Seminars in Cancer Biology"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Abou El Qassim, L., Le Guillou, S., and Royo, L.J. (2022). Variation of miRNA content in cow raw milk depending on the dairy production system. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms231911681"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"102684","DOI":"10.1016\/j.redox.2023.102684","article-title":"miR-484 mediates oxidative stress-induced ovarian dysfunction and promotes granulosa cell apoptosis via SESN2 downregulation","volume":"62","author":"Wang","year":"2023","journal-title":"Redox Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1186\/s12929-024-01085-8","article-title":"Secretome from estrogen-responding human placenta-derived mesenchymal stem cells rescues ovarian function and circadian rhythm in mice with cyclophosphamide-induced primary ovarian insufficiency","volume":"31","author":"Le","year":"2024","journal-title":"J. Biomed. Sci."},{"key":"ref_13","first-page":"1","article-title":"Corpus luteum presence in the bovine ovary increase intrafollicular progesterone concentration: Consequences in follicular cells gene expression and follicular fluid small extracellular vesicles miRNA contents","volume":"17","author":"Rosa","year":"2024","journal-title":"J. Ovarian Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"111765","DOI":"10.1016\/j.mce.2022.111765","article-title":"Totipotency of miR-184 in porcine granulosa cells","volume":"558","author":"Shi","year":"2022","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Liu, J., Ning, C., Zhang, J., Xu, S., Wu, J., Tao, C., Ma, F., Chen, Q., and Pan, Z. (2023). Comparative miRNA expression profile analysis of porcine ovarian follicles: New insights into the initiation mechanism of follicular atresia. Front. Genet., 14.","DOI":"10.3389\/fgene.2023.1338411"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"108441","DOI":"10.1016\/j.reprotox.2023.108441","article-title":"Effects of microcystin-LR on mammalian ovaries","volume":"120","author":"Graceli","year":"2023","journal-title":"Reprod. Toxicol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"RP93172","DOI":"10.7554\/eLife.93172.3","article-title":"Exceptional longevity of mammalian ovarian and oocyte macromolecules throughout the reproductive lifespan","volume":"13","author":"Velez","year":"2024","journal-title":"eLife"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"194","DOI":"10.1186\/s10020-024-00971-z","article-title":"Dihydroberberine alleviates Th17\/Treg imbalance in premature ovarian insufficiency mice via inhibiting Rheb\/mTOR signaling","volume":"30","author":"Deng","year":"2024","journal-title":"Mol. Med."},{"key":"ref_19","first-page":"685","article-title":"Radiosensitivity of human ovarian cancer cells is enhanced by pseudolaric acid B due to the inhibition of the Ras\/Raf\/ERK signaling pathway","volume":"15","author":"Song","year":"2018","journal-title":"Exp. Ther. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.ydbio.2024.02.009","article-title":"Ras\/MAPK signaling mediates adipose tissue control of ovarian germline survival and ovulation in Drosophila melanogaster","volume":"510","author":"Bradshaw","year":"2024","journal-title":"Dev. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1038\/s41420-025-02531-8","article-title":"Single cell RNA sequencing reveals the role of local renin-angiotensin system in regulating ovarian physiological cycle and promoting PCOS","volume":"11","author":"Wei","year":"2025","journal-title":"Cell Death Discov."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Billhaq, D.H., and Lee, S. (2021). The role of the guanosine nucleotide-binding protein in the corpus luteum. Animals, 11.","DOI":"10.3390\/ani11061524"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"938","DOI":"10.1126\/science.1171396","article-title":"MAPK3\/1 (ERK1\/2) in ovarian granulosa cells are essential for female fertility","volume":"324","author":"Fan","year":"2009","journal-title":"Science"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"3522","DOI":"10.1038\/onc.2011.70","article-title":"Molecular and functional characteristics of ovarian surface epithelial cells transformed by KrasG12D and loss of Pten in a mouse model in vivo","volume":"30","author":"Mullany","year":"2011","journal-title":"Oncogene"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1093\/humupd\/dmy047","article-title":"Neurotrophins and glial cell line-derived neurotrophic factor in the ovary: Physiological and pathophysiological implications","volume":"25","author":"Chang","year":"2019","journal-title":"Hum. Reprod. Update"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e2521253123","DOI":"10.1073\/pnas.2521253123","article-title":"The Nemp1\u2013Nesprin complex mediates cellular responses to matrix mechanics","volume":"123","author":"Ganguly","year":"2026","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1186\/s40851-020-00157-5","article-title":"Cellular and molecular aspects of oocyte maturation and fertilization: A perspective from the actin cytoskeleton","volume":"6","author":"Santella","year":"2020","journal-title":"Zool. Lett."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"gaad010","DOI":"10.1093\/molehr\/gaad010","article-title":"Semaphorin 4C regulates ovarian steroidogenesis through RHOA\/ROCK1-mediated actin cytoskeleton rearrangement","volume":"29","author":"Chen","year":"2023","journal-title":"Mol. Hum. Reprod."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Tan, W., Dai, F., Yang, D., Deng, Z., Gu, R., Zhao, X., and Cheng, Y. (2022). MiR-93-5p promotes granulosa cell apoptosis and ferroptosis by the NF-kB signaling pathway in polycystic ovary syndrome. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.967151"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1008","DOI":"10.7150\/ijbs.68028","article-title":"Mir-484 contributes to diminished ovarian reserve by regulating granulosa cell function via YAP1-mediated mitochondrial function and apoptosis","volume":"18","author":"Li","year":"2022","journal-title":"Int. J. Biol. Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4263","DOI":"10.1007\/s00210-023-02895-7","article-title":"The effect of ellagic acid on sex hormones and miRNA-21 expression in rats with polycystic ovary syndrome","volume":"397","author":"Khoshvaghti","year":"2023","journal-title":"Naunyn-Schmiedeberg\u2019s Arch. Pharmacol."},{"key":"ref_32","unstructured":"Tscherner, A. (2017). MicroRNA Expression and Regulation in the Cumulus-Oocyte Complex and Preimplantation Embryo. [Ph.D. Thesis, University of Guelph]."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Gorczyca, G., Wartalski, K., Romek, M., Samiec, M., and Duda, M. (2022). The Molecular Quality and Mitochondrial Activity of Porcine Cumulus\u2013Oocyte Complexes Are Affected by Their Exposure to Three Endocrine-Active Compounds under 3D In Vitro Maturation Conditions. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23094572"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Gu, B., Wang, A., Yu, X., Li, Y., Cong, Y., and Jiang, H. (2025). Integrated Whole-Transcriptome Analysis to Elucidate the Core Regulatory Network of circRNA Involved in Ovarian Development and Reproductive Capacity Differences in Sheep: CircRNA2058-miR-9226-5p-MET Axis. Animals, 15.","DOI":"10.20944\/preprints202509.0351.v1"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1088","DOI":"10.24272\/j.issn.2095-8137.2024.037","article-title":"Single nucleus\/cell RNA-seq of the chicken hypothalamic-pituitary-ovarian axis offers new insights into the molecular regulatory mechanisms of ovarian development","volume":"45","author":"Leng","year":"2024","journal-title":"Zool. Res."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Ren, W., Zhou, J., Zhu, J., Zhang, J., Zhao, X., and Yao, X. (2025). Exploring the Abnormal Characteristics of the Ovaries During the Estrus Period of Kazakh Horses Based on Single-Cell Transcriptome Technology. Biology, 14.","DOI":"10.3390\/biology14101351"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"257","DOI":"10.21451\/1984-3143-AR864","article-title":"Dynamics and role of MicroRNAs during mammalian follicular development","volume":"13","author":"Gebremedhn","year":"2018","journal-title":"Anim. Reprod."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/14\/2197\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T01:19:50Z","timestamp":1784164790000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/14\/2197"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,15]]},"references-count":37,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["ani16142197"],"URL":"https:\/\/doi.org\/10.3390\/ani16142197","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,15]]}}}