Validation of Selected MicroRNA Transcriptome Data in the Bovine Corpus Luteum during Early Pregnancy by RT-qPCR

Abstract

In cattle, the corpus luteum (CL) is pivotal in maintaining early pregnancy by secreting progesterone. To establish pregnancy, the conceptus produces interferon-τ, preventing luteolysis and initiating the transformation of the CL spurium into a CL verum. Although this transformation is tightly regulated, limited data are available on the expression of microRNAs (miRNAs) during and after this process. To address this gap, we re-analyzed previously published RNA-Seq data of CL from pregnant cows and regressed CL from non-pregnant cows. This analysis identified 44 differentially expressed miRNAs. From this pool, three miRNAs-bta-miR-222-3p, bta-miR-29c, and bta-miR-2411-3p-were randomly selected for relative quantification. Using bovine ovaries (n = 14) obtained from an abattoir, total RNA (including miRNAs) was extracted and converted to cDNA for RT-qPCR. The results revealed that bta-miR-222-3p was downregulated (p = 0.016) in pregnant females compared to non-pregnant cows with regressed CL. However, no differences in miRNA expression were observed between CL of pregnant and non-pregnant cows for bta-miR-29c (p > 0.32) or bta-miR-2411-3p (p > 0.60). In silico prediction approaches indicated that these miRNAs are involved in pathways regulating pregnancy maintenance, such as the VEGF- and FoxO-signaling pathways. Additionally, their biogenesis is regulated by GABPA and E2F4 transcription factors. The validation of selected miRNA expression in the CL during pregnancy by RT-qPCR provides novel insights that could potentially lead to the identification of biomarkers related to CL physiology and pregnancy outcome.

Keywords: NGS; RT-qPCR; cattle; corpus albicans; corpus luteum; microRNA; pregnancy.

Figure 1

The expression of miRNAs in the pregnant corpus luteum (CL) regressed CL. The RT-qPCR fold change ratio was measured using the 2^−ddct from fourteen CL (seven from each group). Comparison of mean values was performed using Student’s t-test on the dct values, and the p-values for each pair-wise comparison are indicated for each miRNA. Means were considered statistically different at p < 0.05.

Figure 2

Enrichment results of hsa-miR-222-3p and hsa-miR-29c-3p for the categories of the KEGG database and experimentally validated miRNA–target interactions (MTIs). Rows: enrichment results for the targets of the two miRNAs; columns: all KEGG pathways that are significant for the different miRNAs. The absence of bta-miR-2411-3p on the heat map indicates that no enrichment was observed for this miRNA. The color of each field represents the significance of the association between the miRNA and the target pathway, depicted as the −log(p-value). Darker colors indicate more significant associations between the miRNA and the target pathway.

Figure 3

The molecular function and protein class gene ontology (GO). (a) For molecular function, the GO term “binding” represented 51% and the GO term “molecular function regulator” represented 49% of hits against the total number of genes. (b) For protein class, the GO terms “nucleic acid metabolism protein” and “gene-specific transcriptional regulator” represented 17% and 83%, respectively, of hits against the total number of genes.

Figure 4

A graphical representation of binding sites for the transcription factors (TFs) overlapping transcription start sites (TSSs) of miR-222 within chromosome X as predicted by DIANA-miRGen ^V4.