Evaluation of circulating miRNAs during late pregnancy in the mare

MicroRNAs (miRNAs) are small, non-coding RNAs which are produced throughout the body. Individual tissues tend to have a specific expression profile and excrete many of these miRNAs into circulation. These circulating miRNAs may be diagnostically valuable biomarkers for assessing the presence of disease while minimizing invasive testing. In women, numerous circulating miRNAs have been identified which change significantly during pregnancy-related complications (e.g. chorioamnionitis, eclampsia, recurrent pregnancy loss); however, no prior work has been done in this area in the horse. To identify pregnancy-specific miRNAs, we collected serial whole blood samples in pregnant mares at 8, 9, 10 m of gestation and post-partum, as well as from non-pregnant (diestrous) mares. In total, we evaluated a panel of 178 miRNAs using qPCR, eventually identifying five miRNAs of interest. One miRNA (miR-374b) was differentially regulated through late gestation and four miRNAs (miR-454, miR-133b, miR-486-5p and miR-204b) were differentially regulated between the pregnant and non-pregnant samples. We were able to identify putative targets for the differentially regulated miRNAs using two separate target prediction programs, miRDB and Ingenuity Pathway Analysis. The targets for the miRNAs differentially regulated during pregnancy were predicted to be involved in signaling pathways such as the STAT3 pathway and PI3/AKT signaling pathway, as well as more endocrine-based pathways, including the GnRH, prolactin and insulin signaling pathways. In summary, this study provides novel information about the changes occurring in circulating miRNAs during normal pregnancy, as well as attempting to predict the biological effects induced by these miRNAs.

微小RNA（MicroRNAs，以下简称miRNAs）是一类广泛分布于全身的小型非编码RNA。各组织通常具有独特的表达谱，并可将大量此类miRNAs释放至循环系统中。此类循环miRNAs或可作为极具临床诊断价值的生物标志物，在减少侵入性检测的同时辅助疾病筛查。在人类女性中，已有研究证实诸多循环miRNAs在妊娠相关并发症（如绒毛膜羊膜炎、子痫、复发性流产）发生时会出现显著表达变化，但目前尚无针对马匹该领域的相关研究。为筛选妊娠特异性miRNAs，本研究采集了妊娠母马在妊娠8、9、10个月及产后的系列全血样本，同时采集了非妊娠（间情期）母马的全血样本。本研究通过实时荧光定量PCR（qPCR）共检测了178个miRNAs的表达水平，最终筛选出5个目标miRNAs。其中miR-374b在妊娠晚期呈现差异表达调控，而miR-454、miR-133b、miR-486-5p及miR-204b这4个miRNAs在妊娠与非妊娠母马样本中呈现差异表达调控。本研究借助miRDB及Ingenuity Pathway Analysis两款靶基因预测工具，成功预测出上述差异表达调控miRNAs的潜在靶基因。经预测，妊娠过程中差异表达的miRNAs其靶基因主要参与STAT3通路、PI3/AKT信号通路等信号通路，同时也涉及促性腺激素释放激素（GnRH）、催乳素及胰岛素等内分泌相关信号通路。综上，本研究首次揭示了正常妊娠过程中循环miRNAs的表达变化特征，并对这些miRNAs所介导的生物学效应进行了预测分析。