Abstract
Equine chromosome 24 microRNA cluster (C24MC), the ortholog of human C14MC, is a pregnancy-related miRNA cluster. This cluster is believed to be implicated in embryonic, fetal, and placental development. The current study aimed to characterize the expression profile of this cluster in maternal circulation throughout equine gestation. The expression profile of miRNAs belonging to this cluster was analyzed in the serum of non-pregnant (diestrus), pregnant (25 d, 45 d, 4 mo, 6 mo, 10 mo), and postpartum mares. Among the miRNAs examined, 11 miRNAs were differentially expressed across the analyzed time-points. Four of these miRNAs (eca-miR-1247-3p, eca-miR-134-5p, eca-miR-382-5p, and eca-miR-433-3p) were found to be enriched in the serum of pregnant mares at Day 25 relative to non-pregnant mares. To further assess the accuracy of these miRNAs in differentiating pregnant (25 d) from non-pregnant mares, receiver operating characteristic (ROC) analysis was performed for each of these miRNAs, revealing that eca-miR-1247-3p and eca-miR-134-5p had the highest accuracy (AUCROC = 0.92 and 0.91, respectively; p < 0.05). Moreover, eca-miR-1247-3p, eca-miR-134-5p, eca-miR-409-3p, and eca-miR-379-5p were enriched in the serum of Day 45 pregnant mares. Among those miRNAs, eca-miR-1247-3p and eca-miR-409-3p retained the highest accuracy as shown by ROC analysis. GO analysis revealed that these miRNAs are mainly implicated in nervous system development as well as organ development. Using in situ hybridization, we localized eca-miR-409-3p in the developing embryo (25 d) and extra-embryonic membranes (25 and 45 d). In conclusion, the present study is the first to elucidate the circulating maternal profile of C24MC-associated miRNAs throughout pregnancy and to suggest that serum eca-miR-1247-3p, eca-miR-134-5p, and eca-miR-409-3p could be used as pregnancy-specific markers during early gestation (25 and 45 d). Overall, the high abundance of these embryo-derived miRNAs in the maternal circulation suggests an embryo-maternal communication during the equine early pregnancy.
Highlights
MicroRNAs are a class of small non-coding RNA that post-transcriptionally regulate protein-coding mRNAs [1]
We demonstrated that chromosome 24 microRNA cluster (C24MC)-associated miRNAs were upregulated in chorioallantoic membrane (CAM) during the early stages of equine pregnancy, followed by a downregulation later in gestation [29]
We hypothesized that equine C24MC-associated miRNAs, the ortholog of human chromosome 14 miRNA cluster (C14MC), will have a differential expression pattern in maternal circulation throughout gestation
Summary
MicroRNAs (miRNAs) are a class of small non-coding RNA that post-transcriptionally regulate protein-coding mRNAs [1]. Throughout the genome, a group of two or more miRNAs are transcribed from physically adjacent miRNA genes (within 10 kb), which form a cluster [9,10] These miRNA clusters have been found to be exclusively or preferentially expressed in a tissue-specific manner [11]. There has been growing interest in pregnancy-related and/or placenta-specific miRNAs in mammals [7,21,30,31,32,33] This interest led to the identification of several circulating miRNAs as biomarkers for early pregnancy diagnosis [34], prediction and/or diagnosis of embryonic loss [35,36], ectopic pregnancy [37,38], pre-eclampsia [39,40], intrauterine growth restriction [40,41], placental infection [41], and preterm labor [41,42]. We hypothesized that equine C24MC-associated miRNAs, the ortholog of human C14MC, will have a differential expression pattern in maternal circulation throughout gestation. This study was designed to evaluate the expression profile of equine C24MC-associated miRNAs in the serum of non-pregnant (diestrus), pregnant (25 days, 45 days, 4 months, 6 months, 10 months) and postpartum mares
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