CircRNAs Related to Breast Muscle Development and Their Interaction Regulatory Network in Gushi Chicken

Abstract

Circular RNAs (circRNAs) play a significant regulatory role during skeletal muscle development. To identify circRNAs during postnatal skeletal muscle development in chickens, we constructed 12 cDNA libraries from breast muscle tissues of Chinese Gushi chickens at 6, 14, 22, and 30 weeks and performed RNA sequencing. In total, 2112 circRNAs were identified, and among them 79.92% were derived from exons. CircRNAs are distributed on all chromosomes of chickens, especially chromosomes 1-9 and Z. Bioinformatics analysis showed that each circRNA had an average of 38 miRNA binding sites, 61.32% of which have internal ribosomal entry site (IRES) elements. Furthermore, in total 543 differentially expressed circRNAs (DE-circRNAs) were identified. Functional enrichment analysis revealed that DE-circRNAs source genes are engaged in biological processes and muscle development-related pathways; for example, cell differentiation, sarcomere, and myofibril formation, mTOR signaling pathway, and TGF-β signaling pathway, etc. We also established a competitive endogenous RNA (ceRNA) regulatory network associated with skeletal muscle development. The results in this report indicate that circRNAs can mediate the development of chicken skeletal muscle by means of a complex ceRNA network among circRNAs, miRNAs, genes, and pathways. The findings of this study might help increase the number of known circRNAs in skeletal muscle tissue and offer a worthwhile resource to further investigate the function of circRNAs in chicken skeletal muscle development.

Keywords: Gushi chickens; ceRNA; circular RNAs; regulatory network; skeletal muscle.

Figure 1

Validation of eight circRNAs. (A) Electropherogram of PCR products. M: marker. (B) Sanger sequencing confirmed the head-to-tail junction of eight circRNAs. Black lines indicate the location of back splicing. The region marked in red (or black) is the terminal (or start) sequence of the circRNA (junction point). (C) Resistance of circRNAs to RNase digestion was detected by qRT-PCR. Control was β-actin. Data are presented as mean ± SEM (n = 3).

Figure 2

Expression analysis of circRNAs. (A) Density distributions of circRNAs TPM values in various libraries. (B) Boxplots of TPM values of circRNAs in various libraries. (C) DE-circRNAs in six comparison groups. The abscissa represents different comparison groups, and the ordinate represents the number. Green bars represent the number of down-regulated DE-circRNAs, while red bars represent the number of up-regulated DE-circRNAs. (D) Hierarchical clustering of DE-circRNAs. (E) K-means cluster plot of DE-circRNAs. (F) Venn diagram of the DE-circRNAs in different comparison groups.

Figure 3

GO and KEGG functional enrichment analysis of source genes of DE-circRNAs. (A) The y-axis is the GO term at the next level of the three GO categories, and the x-axis is the number of source genes annotated for that term. (B) The y-axis indicates the pathway name, the x-axis indicates the p-value, the size of the dots indicates the number of source genes in the pathway, and the color of the dots corresponds to the different −log10 (q-value) ranges.

Figure 4

Skeletal-muscle-development-related pathways involved in differentially expressed mRNAs and DE-circRNAs.

Figure 5

CircRNA-miRNA interaction network associated with skeletal muscle development. miRNAs and CircRNAs are expressed as triangles and circles, respectively.

Figure 6

CircRNA-miRNA-gene interaction network related to skeletal muscle development. The circle, inverted arrows, and triangles represent circRNAs, miRNAs, and mRNAs, respectively.

Figure 7

CircRNA-miRNA-gene interaction network related to cell growth. The circle, inverted arrows, and triangles represent circRNAs, miRNAs, and mRNAs, respectively.