Defining age- and lactocrine-sensitive elements of the neonatal porcine uterine microRNA-mRNA interactome

Abstract

Factors delivered to offspring in colostrum within 2 days of birth support neonatal porcine uterine development. The uterine mRNA transcriptome is affected by age and nursing during this period. Whether uterine microRNA (miRNA) expression is affected similarly is unknown. Objectives were to (1) determine effects of age and nursing on porcine uterine miRNA expression between birth and postnatal day (PND) 2 using miRNA sequencing (miRNAseq) and; (2) define affected miRNA–mRNA interactions and associated biological processes using integrated target prediction analysis. At birth (PND 0), gilts were euthanized, nursed ad libitum, or gavage-fed milk replacer for 48 h. Uteri were collected at birth or 50 h postnatal. MicroRNAseq data were validated using quantitative real-time PCR. Targets were predicted using an established mRNA database generated from the same tissues. For PND 2 versus PND 0 comparisons, 31 differentially expressed (DE) miRNAs were identified for nursed, and 42 DE miRNAs were identified for replacer-fed gilts. Six DE miRNAs were identified for nursed versus replacer-fed gilts on PND 2. Target prediction for inversely correlated DE miRNA–mRNA pairings indicated 20 miRNAs targeting 251 mRNAs in nursed, versus 29 miRNAs targeting 585 mRNAs in replacer-fed gilts for PND 2 versus PND 0 comparisons, and 5 miRNAs targeting 81 mRNAs for nursed versus replacer-fed gilts on PND 2. Biological processes predicted to be affected by age and nursing included cell-to-cell signaling, cell morphology, and tissue morphology. Results indicate novel age- and lactocrine-sensitive miRNA–mRNA relationships associated with porcine neonatal uterine development between birth and PND 2

Keywords: neonate; uterus; pig; microRNA; transcriptome; lactocrine.

Figure 1.

(A) Principal component analysis (PCA) plot and (B) Spearman correlation heat map from miRNAseq analysis. (A) PCA plot of pooled neonatal porcine uterine samples showing the clustering of expressed miRNAs for PND 0 (green), PND 2N (blue), and PND 2R (purple). (B) Heat map of pairwise correlations between pooled samples based on the Spearman correlation coefficients. Light green represents highest correlations and bright red represents lowest correlations.

Figure 2.

Volcano plots (left) demonstrating differences in uterine expression of miRNAs between (A) PND 2N versus PND 0, (B) PND 2R versus PND 0, and (C) PND 2N versus PND 2R. Upregulated miRNAs are indicated in red (fold change ≥ 2) and downregulated miRNAs are denoted in green. Black indicates miRNAs that did not change between groups. The horizontal line indicates P = 0.05. For each comparison, the total number of differentially expressed miRNAs is given (Δ value). The number of miRNAs that were downregulated (green) or upregulated (red) are shown. Results of qPCR validation (right) for six miRNAs identified by miRNAseq for (D) PND 2N versus PND 0; (E) PND 2R versus PND 0; (F) PND 2N versus PND 2R. White bars indicate miRNAseq fold change; black bars indicate qPCR fold change. An overall positive correlation between miRNAseq and qPCR results was identified (r = 0.76, P < 0.01).

Figure 3.

Venn diagram illustrating unique and overlapping differential miRNA expression domains for the three group comparisons [PND 2N versus PND 0 (green); PND 2R versus PND 0 (purple); PND 2N versus PND 2R (blue)]. Values indicate the number of differentially expressed miRNAs associated with unique and overlapping domains. Differentially expressed miRNAs determined to be unique to each comparison are listed in descending order for absolute fold change (AFC > 2-fold; red = upregulated, green = downregulated). Superscript 1 indicates miRNAs with same name found at different genomic loci.

Figure 4.

High-throughput sequencing workflow and output. Uterine tissues were obtained from gilts (1) at birth on PND 0, before nursing; (2) after nursing ad libitum from birth through 48 h of age (PND 2N); or (3) after gavage feeding a commercial pig milk replacer for 48 h (PND 2R). Total uterine RNA was isolated and both small RNAs and mRNAs were sequenced as described in section Materials and Methods. Asterisk indicates mRNAseq data for uterine mRNAs as described in Rahman et al [8]. The numbers of differentially expressed miRNAs and mRNAs for each comparison are presented. Integrated target prediction analysis was conducted using IPA as described in section Materials and Methods. The numbers of differentially expressed miRNAs targeting differentially expressed mRNAs are presented for each comparison. Individual miRNAs targeting mRNAs are listed (bottom; absolute fold change > 2; red = upregulated, green = downregulated).

Figure 5.

Selected functional annotation categories for differentially expressed transcripts (miRNAs and mRNAs) associated with miRNA–mRNA interactions between (A) PND 2N versus PND 0; (B) PND 2R versus PND 0; and (C) PND 2N versus PND 2R as identified by IPA. Values within parentheses indicate the number of annotated mRNAs targeted by miRNAs that are involved in the corresponding functional term. Enrichment scores were calculated by taking the geometric mean of the P-values associated with differentially expressed transcripts (in –log₁₀ scale). Dashed vertical line indicates P = 0.05.

Figure 6.

Integrated uterine miRNA–mRNA interactomes illustrating effects of (A) PND 2N versus PND 0; (B) PND 2R versus PND 0; and (C) PND 2N versus PND 2R on cell-to-cell signaling and interaction. Red denotes increased and green denotes decreased transcript expression (P < 0.05). Color intensity indicates degree of change. Inset provides an opportunity to study detailed relationships within the interactome network. IPA legend key (left): (1) cytokine/growth factor; (2) enzyme; (3) G-protein coupled receptor; (4) growth factor; (5) ion channel; (6) kinase; (7) peptidase; (8) phosphatase; (9) transcription regulator; (10) transmembrane receptor; (11) transporter; (12) other; and (13) mature miRNA.