Evaluation of circulating miRNAs during late pregnancy in the mare

<div><p>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.</p></div

Differential expression of miR-374b through late gestation.

<p>Expression of circulating miRNAs was analyzed using quantitative PCR through late gestation (8, 9 or 10 months) and post-partum (PP). One miRNA, miR-374b, was found to be differentially regulated. Data shown represent the ΔCt value (dCt), calculated as ΔCt = Ct_(target)−Ct_{(mean of sample)}, with each dot representative of a separate sample. A lower ΔCt value indicates a higher expression level; for each 1-point reduction in ΔCt, an approximately 2-fold increase in expression can be assumed. Significantly different samples are indicated by varying superscripts.</p

Differential expression of circulating miRNAs in pregnant mares.

<p>Expression of circulating miRNAs was analyzed using quantitative PCR to identify miRNAs with significantly different expression during pregnancy. Samples were grouped into pregnant (8, 9, 10 m gestation, postpartum (PP)) or non-pregnant (diestrus) samples and analyzed by one-way ANOVA corrected for false discovery rate (Benjamini-Hochberg; P < 0.05), with post-hoc analysis performed by student’s t-test (P < 0.1). Data were normalized by ΔCt = Ct_(target)−Ct_{(mean of the sample)}, with each point representing the ΔCt for an individual mare. A lower ΔCt value indicates a higher expression level; for each 1-point reduction in ΔCt, an approximately 2-fold increase in expression can be assumed. Significantly different samples are indicated by varying superscripts.</p

Top twenty pathways predicted for the miRNA differentially regulated through late gestation (miR-374b).

<p>Top twenty pathways predicted for the miRNA differentially regulated through late gestation (miR-374b).</p

Top twenty pathways predicted for miRNAs differentially regulated during pregnancy (miR-133b, miR-204b, miR-454, miR-486-5p).

<p>Top twenty pathways predicted for miRNAs differentially regulated during pregnancy (miR-133b, miR-204b, miR-454, miR-486-5p).</p