College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, 471023, Henan, China.
Sci Rep. 2024 Nov 9;14(1):27353. doi: 10.1038/s41598-024-79062-2.
Our study concentrated on the expression of miRNA-375 in the hypothalamic-pituitary-gonadal axis of female Hu sheep. The investigation involved cloning the precursor sequence of miR-NA-375, followed by comparison with database entries and subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In our approach, we obtained ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus, and pineal gland from fertile but nonpregnant Hu ewes. MiRNA extraction kit was used to extract miRNA from the above eight tissues. Real-time fluorescent quantitative polymerase chain reaction was used to evaluate the role of miR-375 in the hy-pothalamic-pituitary-gonadal axis. The results of miR-375 precursor sequence cloning were compared with those of Anopheles gambiae, Apis mellifera, Bos taurus, Drosophila melanogaster, Danio rerio, Fugu rubripes, Gallus gallus, Homo sapiens, Monodelphis domestica, Macaca mulatta, Mus musculus, Pan troglodytes, Rattus norvegicus, Tetraodon nigroviridis, Xenopus tropicalis miR-375 in miRBase database. It was found that oar-miR-375 was highly conserved. Notably, miR-375 expression in the pineal gland was significantly higher (p < 0.01) than that in the ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus. The study also involved predicting miR-375 target genes. GO and KEGG enrichment analyses of these predicted target genes revealed that miR-375 is involved in 182 biological processes, affects 186 cellular components, and participates in 184 molecular functions. In terms of pathway enrichment, miR-375 was linked to nine pathways, including the Hippo, Wnt, and mTOR signaling pathways. This study has validated the interaction between miR-375 and its target gene FZD4, which can be recognized and bound to produce effects. These findings lead to the inference that miR-375 may play a crucial regulatory role in sheep reproduction through the Hippo pathway and Wnt pathway, laying a foundation for further exploration of miR-375's role in this domain.
我们的研究集中在雌性湖羊下丘脑-垂体-性腺轴中 miRNA-375 的表达上。研究包括克隆 miRNA-375 的前体序列，然后与数据库条目进行比较，随后进行基因本体 (GO) 和京都基因与基因组百科全书 (KEGG) 富集分析。在我们的方法中，我们从有生育能力但未怀孕的湖羊中获得了卵巢、丘脑、小脑、大脑、子宫、垂体、下丘脑和松果体。我们使用 miRNA 提取试剂盒从上述 8 种组织中提取 miRNA。实时荧光定量聚合酶链反应用于评估 miR-375 在下丘脑-垂体-性腺轴中的作用。miR-375 前体序列克隆的结果与疟蚊、蜜蜂、牛、黑腹果蝇、斑马鱼、红鳍东方鲀、鸡、智人、短尾猴、猕猴、小家鼠、黑猩猩、大鼠、绿斑河豚、爪蟾在 miRBase 数据库中的 miR-375 进行了比较。结果发现 oar-miR-375 高度保守。值得注意的是，松果体中的 miR-375 表达明显高于卵巢、丘脑、小脑、大脑、子宫、垂体、下丘脑（p<0.01）。研究还涉及预测 miR-375 的靶基因。这些预测靶基因的 GO 和 KEGG 富集分析表明，miR-375 参与了 182 个生物学过程，影响了 186 个细胞成分，并参与了 184 个分子功能。就途径富集而言，miR-375 与 Hippo、Wnt 和 mTOR 信号通路等 9 条途径有关。本研究验证了 miR-375 与其靶基因 FZD4 之间的相互作用，它们可以被识别并结合产生作用。这些发现导致推断 miR-375 可能通过 Hippo 途径和 Wnt 途径在绵羊生殖中发挥关键调节作用，为进一步探索 miR-375 在这一领域的作用奠定了基础。
