Genome-wide analysis of circular RNA-mediated ceRNA regulation in porcine skeletal muscle development

Abstract

Background: As a diverse and abundant class of endogenous RNAs, circular RNAs (circRNAs) participate in various biological processes including cell proliferation and apoptosis. Nevertheless, few researchers have investigated the role of circRNAs in muscle development in cultivated pigs.

Results: In this study, we used RNA-seq to construct circRNA expression profiles in skeletal muscle of Jinfen White pigs at the age of 1, 90, and 180 days. Among the 16,990 identified circRNAs, 584 circRNAs were differentially expressed. Moreover, the enrichment analysis of DE circRNA host genes showed that they were mainly involved in muscle contraction, muscle organ development and muscle system processes, as well as AMPK and cAMP-related signal pathways. We also constructed a circRNA-miRNA-mRNA co-expression network to find key circRNAs which many involved in the regulation of porcine skeletal muscle development through the competitive endogenous RNA (ceRNA) mechanism. It is noteworthy that circ_0018595/miR-1343/PGM1 axis may play a regulatory role in the development of porcine skeletal muscle.

Conclusions: This study identified the circRNAs and present the circRNA expression profile in the development of pigs, revealed that DE circRNA host genes participate in different cell fates and enriched the porcine ceRNA network. Thus, this work will become a valuable resource for further in-depth study of the regulatory mechanism of circRNA in the development of porcine skeletal muscle.

Keywords: Pig; RNA-seq; Skeletal muscle; ceRNA; circRNA.

Fig. 1

Features of identified circRNAs. A Chromosomal distribution of circRNAs from all samples. B Source distribution of circRNAs for each sample. C Length distribution of circRNAs for all samples

Fig. 2

Expression and number of the DE circRNAs at different developmental stages. A Expression heatmap of DE circRNAs from samples at three different growth stages, with rows showing circRNAs and columns showing tissues. B The number of differentially expressed circRNAs in each comparison including JFW_90d vs. JFW_1d, JFW_180d vs. JFW_1d, and JFW_180d vs. JFW_90d groups. C Venn diagram showing the number of shared DE circRNAs among the three pairwise comparisons

Fig. 3

Validation of a subset of the putative circRNAs. A Validation of differential expressed circRNAs using qRT-PCR. B Representative examples of PCR products purified and sequenced to confirm circRNA junction sequences

Fig. 4

Functional analysis of the DE circRNAs-host genes. A Top 25 GO terms enriched for DE circRNA host genes from the three groups of pairwise comparisons. The y-axis presents the number of DE circRNA host genes in a category, while the x-axis shows the specific GO term. The red histogram represents biological processes, green histogram represents cellular components, and the blue histogram represents molecular functions. B Top 25 KEGG pathways enriched for DE circRNAs host genes from the three pairwise comparisons groups. The x-axis represents the rich factor (number of DE circRNAs enriched in the pathway / number of annotated pathway genes), while the y-axis shows the specific pathways

Fig. 5

The parts of constructed circRNA–miRNA–mRNA (ceRNA) network. The ceRNA networks showing the top 20 DE circRNAs with the most significant upregulation (A) or downregulation (B) across the three muscle developmental stages in Jinfen White pigs

Fig. 6

Characterization of circ_0018595. A Verification of circ_0018595 as a circRNA. B qRT-PCR results showing the resistance of circ_0018595 to RNase R digestion. C Expression levels of circ_0018595 in different tissues of Jinfen White pigs. D Expression changes of circ_0018595 in Jinfen White pigs at 1 d, 90 d, and 180 d. E Nuclear and cytoplasmic levels of circ_0018595 in porcine skeletal muscle satellite cells. F Modeled potential interactions between circ_0018595, PGM1, and miR-1343 generated using RNAhybrid. Note: In panels (C) and (D), uppercase letters are used to indicate extremely significant differences