Dynamic Expression and Regulatory Network of Circular RNA for Abdominal Preadipocytes Differentiation in Chicken (Gallus gallus)

Abstract

Circular RNA (circRNA), as a novel endogenous biomolecule, has been emergingly demonstrated to play crucial roles in mammalian lipid metabolism and obesity. However, little is known about their genome-wide identification, expression profile, and function in chicken adipogenesis. In present study, the adipogenic differentiation of chicken abdominal preadipocyte was successfully induced, and the regulatory functional circRNAs in chicken adipogenesis were identified from abdominal adipocytes at different differentiation stages using Ribo-Zero RNA-seq. A total of 1,068 circRNA candidates were identified and mostly derived from exons. Of these, 111 differentially expressed circRNAs (DE-circRNAs) were detected, characterized by stage-specific expression, and enriched in several lipid-related pathways, such as Hippo signaling pathway, mTOR signaling pathway. Through weighted gene co-expression network analyses (WGCNA) and K-means clustering analyses, two DE-circRNAs, Z:35565770|35568133 and Z:54674624|54755962, were identified as candidate regulatory circRNAs in chicken adipogenic differentiation. Z:35565770|35568133 might compete splicing with its parental gene, ABHD17B, owing to its strictly negative co-expression. We also constructed competing endogenous RNA (ceRNA) network based on DE-circRNA, DE-miRNA, DE-mRNAs, revealing that Z:54674624|54755962 might function as a ceRNA to regulate chicken adipogenic differentiation through the gga-miR-1635-AHR2/IRF1/MGAT3/ABCA1/AADAC and/or the novel_miR_232-STAT5A axis. Translation activity analysis showed that Z:35565770|35568133 and Z:54674624|54755962 have no protein-coding potential. These findings provide valuable evidence for a better understanding of the specific functions and molecular mechanisms of circRNAs underlying avian adipogenesis.

Keywords: abdominal fat; adipogenic differentiation; chicken; circRNA; competing endogenous RNA.

FIGURE 1

Adipogenesis of chicken abdominal preadipocytes at different differentiation stages. (A) Procedure for inducing adipogenic differentiation of abdominal preadipocytes in chicken; (B) Spectrophotometric analysis at the 490 nm absorbance of adipocytic lipid droplet stained by oil red O; (C) Microscopy of oil Red O staining of chicken abdominal adipocytes at 0, 6, 12, 24, 48, 72, 96 and 120 h after differentiation (20X); (D) BODIPY fluorescent staining of chicken abdominal adipocytes at 0, 6, 12, 24, 48, 72, 96 and 120 h after differentiation (20X).

FIGURE 2

Characteristics of circRNAs in chicken abdominal adipocytes during differentiation stages. (A) Venn analysis of adipocyte circRNAs in each differentiation stage; (B) Distribution of genomic regions from where the identified circRNAs were derived; (C) Length distribution of the identified circRNAs; (D) Chromosomal distribution of the identified circRNAs; (E) The numbers of circRNAs derived from per parental gene; (F) Exon numbers of the identified circRNAs.

FIGURE 3

Differentially expressed circRNAs during chicken abdominal adipocytes differentiation stages. (A) Heatmap of differentially expressed circRNAs in four comparisons (A0 vs A12, A12 vs A48, A48 vs A72, A72 vs A120); (B) Histogram of four comparisons of the number of differentially expressed circRNAs; (C) Venn analysis of differentially expressed circRNAs; (D) Volcano map of differentially expressed circRNAs in A0 vs A12, A12 vs A48, A48 vs A72, A72 vs A120. The DE-circRNA with p value < 0.05 and log2foldchange > 5 were marked in red, and DE-circRNA with p value < 0.05 and log2foldchange < −5 were marked in blue.

FIGURE 4

Cyclization and expression validation of circRNAs. (A) An example of circRNA validation. 8:27886180|27889657, derived from exon 19 to exon 22 of DOCK7 gene, was indeed amplified with divergent primers in the cDNA but not gDNA form. Sanger sequencing confirmed the BSJ sites of 8:27886180|27889657. (B) An example of circRNA validation. 5:38248809|38268609, derived from exon 2 to exon 17 of YLPM1 gene, was also indeed amplified with divergent primers in the cDNA but not gDNA form. Sanger sequencing confirmed the BSJ sites of 5:38248809|38268609. (C) Detection of circRNA expression in adipocytes at different differentiation stages using qRT-PCR. All circRNA expression values are shown as fold change values vs that of 0 h group. All data are represented as the mean ± SD (n = 3). *p < 0.05, **p < 0.01.

FIGURE 5

GO annotation and KEGG enrichment analysis of parental genes of DE-circRNAs. (A) Top 20 significantly enriched GO terms of DE-circRNAs in the biological process, cellular component and molecular function; (B) Top 20 KEGG pathways of DE-circRNAs.

FIGURE 6

Clustering of all differentially expressed circRNAs and mRNAs. (A) Heatmap displays the K-means clustering of transformed expression abundances for circRNAs and mRNAs. Yellow means higher expression, and purple means lower expression; (B) Expression patterns of circRNAs and mRNAs in group 1 involving K02, K05, K14, K15 clusters; (C) Expression patterns of circRNAs and mRNAs in group 2 involving K06, K10 clusters; (D) Expression patterns of circRNAs and mRNAs in group 3 involving K09 and K11 clusters; (E) Expression patterns of circRNAs and mRNAs in group 4 involving K04 cluster; (F) Expression patterns of circRNAs and mRNAs in group 5 involving K01, K08, K12 clusters; (G) Expression patterns of circRNAs and mRNAs in group 6 involving K07 cluster; (H) Expression patterns of circRNAs and mRNAs in group 7 involving K03, K13 clusters.

FIGURE 7

WGCNA of all circRNAs and mRNAs identified from chicken abdominal adipocytes over four differentiation stages. (A) Hierarchical cluster tree showing 26 modules of co-expressed genes. Each gene is represented by a leaf in the tree, and each module is represented by a major tree branch. The lower panel shows the modules in designated colors, in which module “grey” refers to unassigned genes; (B) Correlation analysis of module and differentiation stages. The color ranging from blue to red in heatmap indicates higher correlation.

FIGURE 8

Visualization of GS vs MM and expression level in 10 interesting modules. (A) A scatterplot of GS for differentiation stage vs MM in modules; (B) The heatmap and bar plot represent the expression level of genes in modules in 15 samples. The color ranging from green to red in heatmap indicates higher expression levels.

FIGURE 9

Density diagram of correlation between circRNAs and their parental genes. (A) Density diagram of correlation between circRNAs and their parental genes; (B) Density diagram of significant expression correlation between circRNAs and their parental genes (p < 0.05). The red, green and blue line represents the expression correlation of exonic circRNA-parental gene pairs, intronic circRNA-parental gene pairs and intergenic circRNA-parental gene pairs, respectively.

FIGURE 10

The ceRNA regulatory network of circRNA-miRNA-mRNA in chicken abdominal adipocytes from different differentiation stages. The triangle filled with red represents circRNAs, the rectangles filled with blue represents miRNAs, the circles filled with orange and turquoise represents transcription factor and genes, respectively.

FIGURE 11

Translation capacity prediction of circRNAs based on ORF and IRES sequences. (A) Veen analysis of circRNAs with IRES, ORF and ORF crossing the backsplicing junctions; (B) Veen analysis of exonic circRNAs and circRNAs with IRES and ORF, circRNAs with IRES and ORF crossing the backsplicing junctions; (C) Veen analysis of intronic circRNAs and circRNAs with IRES and ORF, circRNAs with IRES and ORF crossing the backsplicing junctions; (D) Veen analysis of intergenic circRNAs and circRNAs with IRES and ORF, circRNAs with IRES and ORF crossing the backsplicing junctions.