Identification and characterization of circRNAs in peri-implantation endometrium between Yorkshire and Erhualian pigs

Abstract

Background: One of the most critical periods for the loss of pig embryos is the 12th day of gestation when implantation begins. Recent studies have shown that non-coding RNAs (ncRNAs) play important regulatory roles during pregnancy. Circular RNAs (circRNAs) are a kind of ubiquitously expressed ncRNAs that can directly regulate the binding proteins or regulate the expression of target genes by adsorbing micro RNAs (miRNA).

Results: We used the Illumina Novaseq6,000 technology to analyze the circRNA expression profile in the endometrium of three Erhualian (EH12) and three Yorkshire (YK12) pigs on day 12 of gestation. Overall, a total of 22,108 circRNAs were identified. Of these, 4051 circRNAs were specific to EH12 and 5889 circRNAs were specific to YK12, indicating a high level of breed specificity. Further analysis showed that there were 641 significant differentially expressed circRNAs (SDEcircRNAs) in EH12 compared with YK12 (FDR < 0.05). Functional enrichment of differential circRNA host genes revealed many pathways and genes associated with reproduction and regulation of embryo development. Network analysis of circRNA-miRNA interactions further supported the idea that circRNAs act as sponges for miRNAs to regulate gene expression. The prediction of differential circRNA binding proteins further explored the potential regulatory pathways of circRNAs. Analysis of SDEcircRNAs suggested a possible reason for the difference in embryo survival between the two breeds at the peri-implantation stage.

Conclusions: Together, these data suggest that circRNAs are abundantly expressed in the endometrium during the peri-implantation period in pigs and are important regulators of related genes. The results of this study will help to further understand the differences in molecular pathways between the two breeds during the critical implantation period of pregnancy, and will help to provide insight into the molecular mechanisms that contribute to the establishment of pregnancy and embryo loss in pigs.

Keywords: CircRNA; Embryo implantation; Endometrium; Erhualian; Peri-Implantation; Yorkshire.

Fig. 1

Comparison of Yorkshire and Erhualian uterus samples. (A) Comparison of uterine morphology between Yorkshire and Erhualian during peri-implantation. PCA (B) and cluster dendrogram (C) characterize the clustering distribution of Erhualian and Yorkshire endometrium samples, respectively

Fig. 2

Characterization of circRNA expression in the endometrium of Yorkshire and Erhualian pigs during the peri-implantation period. (A) Filter Statistics Percentage. (B) spliced reads comparison reference area statistics. (C) Length distribution of circRNAs. (D) Frequency of back-splicing reads of circRNAs identified in endometrial tissues of 3 Yorkshire and 3 Erhualian pigs. (E) The number of circRNAs identified in each sample of Yorkshire and Erhualian endometrium. (F) The genomic origin of porcine circRNAs. (G) Violin plot of relative abundance of circRNAs in Yorkshire and Erhualian endometrial tissue. Data are expressed as log2 changes in RPM. White dots represent medians. (H) Chromosome distribution of the sequences from which circRNAs were derived

Fig. 3

The top 25 circRNAs with the highest content in Yorkshire (A) and Erhualian (B) endometrium. Left axis and histogram: Percentage of each circRNA to total circRNA reads. Right axis and dots: cumulative percentage of circRNA reads

Fig. 4

Expression profiles of DEcircRNAs. (A) Number of unique circRNAs tags between Yorkshire and Erhualian. (B) Histogram of SDEcircRNAs between Yorkshire and Erhualian. (C) Volcano map of SDEcircRNAs. (D) Clustering heatmap of DEcircRNAs.

Fig. 5

Histogram of GO-enriched classification of DEcircRNAs. The data labels on the bar graph are q-values

Fig. 6

KEGG enrichment scatter plot of DEcircRNAs host genes

Fig. 7

Exploration of circRNA mediated ceRNA regulatory network. (A) Illustration of the ceRNA interaction network. Blue nodes represent miRNAs, green nodes represent down-regulated circRNAs (top 25 DEcircRNAs in YK12), and red nodes represent up-regulated circRNAs (top 25 DEcircRNAs in EH12). (B) Sub-network of miRNAs with the abundant interactions

Fig. 8

Prediction of differential circRNAs binding proteins. RBP-binding motifs are enriched in flanking regions (A) and themselves sequences (B) of circRNAs.

Fig. 9

RT-qPCR validation of nine diferentially expressed circRNAs between YK12 and EH12.

Fig. 10

Experimental validation of circRNAs. (A) Sanger sequencing to verify the backsplice junction sequence of circRNA. (B) Fluorescence quantitative PCR showed that the circRNA was RNase R enzyme resistant. *P < 0.05; **P < 0.01