Table_6_miRNA-150_R-1 mediates the HIF-1/ErbB signaling pathway to regulate the adhesion of endometrial epithelial cells in cows experiencing retained placenta.XLSX

Retained placenta (RP) refers to reproductive disorders caused by the failure of fetal membranes to be expelled 12 h after delivery in dairy cows. Postpartum adhesion of the fetal membranes to the uterus causes diseases such as mastitis or endometritis, which threatening the profitability of the dairy industry. Emerging evidence suggests that micro RNAs (miRNAs) play crucial roles in various processes, such as the occurrence and progression of fetal membranes discharge. However, the molecular mechanisms of miRNAs in RP remain unknown. In this study, we performed RNA-sequencing to characterize the expression profiles of mRNAs and miRNAs in caudal vein blood samples of postpartum Holstein cows whose fetal membranes were discharged normally or retained to identify RP-related genes and evaluate their molecular mechanisms. We identified 44 differentially expressed miRNAs (19 upregulated and 25 downregulated) and 706 differentially expressed mRNAs (325 upregulated and 381 downregulated) in the RP group compared to the normal fetal membranes discharge group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed that differentially expressed mRNAs were mainly enriched in the extracellular matrix, cell adhesion, and autoimmunity-related biological processes or pathways. Further analyses using RNA-sequencing, a dual luciferase reporter system, quantitative reverse transcription-PCR, immunofluorescence, and western blotting verified that endothelial PAS domain protein 1 (EPAS1) is regulated by miR-150_R-1 in endometrial epithelial cells. We demonstrated the relationship between EPAS1 and RP and confirmed that EPAS1 is upregulated in the blood and placenta of cows that experience RP. Further, we proposed a model of the miRNA-mRNA negative regulatory network mediated by the HIF-1/ErbB signaling pathway to show its regulatory role in RP.

胎衣不下（Retained placenta, RP）指奶牛分娩后胎膜未能在12小时内排出所引发的生殖系统疾病。产后胎膜黏连于子宫可诱发乳腺炎、子宫内膜炎等病症，对奶牛养殖业的经济效益造成威胁。新近研究表明，微小RNA（micro RNA, miRNA）在包括胎膜排出过程的发生与发展在内的多种生理病理过程中发挥关键调控作用，但miRNA在胎衣不下中的分子机制仍未明确。本研究通过RNA测序（RNA-sequencing），对产后胎膜正常排出与胎衣不下的荷斯坦奶牛尾静脉血液样本中的信使RNA（mRNA）和miRNA表达谱进行表征，以筛选RP相关基因并解析其分子调控机制。与正常胎膜排出组相比，胎衣不下组共鉴定得到44个差异表达miRNA（其中19个上调、25个下调）以及706个差异表达mRNA（其中325个上调、381个下调）。基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析结果显示，差异表达mRNA主要富集于细胞外基质、细胞黏附以及自身免疫相关的生物学过程与信号通路中。本研究进一步结合RNA测序、双荧光素酶报告基因系统、实时定量反转录PCR（quantitative reverse transcription-PCR）、免疫荧光以及蛋白质印迹（western blotting）实验，验证了内皮PAS结构域蛋白1（endothelial PAS domain protein 1, EPAS1）在子宫内膜上皮细胞中受miR-150_R-1调控。本研究阐明了EPAS1与胎衣不下的关联，并证实胎衣不下奶牛的血液与胎盘组织中EPAS1表达上调。此外，本研究提出了由HIF-1/ErbB信号通路介导的miRNA-mRNA负调控网络模型，以阐释其在胎衣不下中的调控作用。