Comparative Transcriptome Analysis Reveals Regulatory Mechanism of Long Non-Coding RNAs during Abdominal Preadipocyte Adipogenic Differentiation in Chickens

Abstract

Long non-coding RNAs (lncRNAs) are implicated in mammalian adipogenesis and obesity. However, their genome-wide distribution, expression profiles, and regulatory mechanisms during chicken adipogenesis remain rarely understood. In the present study, lncRNAs associated with adipogenesis were identified from chicken abdominal adipocytes at multiple differentiation stages using Ribo-Zero RNA-seq. A total of 15,179 lncRNAs were identified and characterized by stage-specific expression patterns. Of these, 840 differentially expressed lncRNAs were detected, and their cis- and trans-target genes were significantly enriched in multiple lipid-related pathways. Through weighted gene co-expression network analysis (WGCNA) and time-series expression profile clustering analysis, 14 key lncRNAs were identified as candidate regulatory lncRNAs in chicken adipogenic differentiation. The cis- and trans-regulatory interactions of key lncRNAs were constructed based on their differentially expressed cis- and trans-target genes, respectively. We also constructed a competing endogenous RNA (ceRNA) network based on the key lncRNAs, differentially expressed miRNAs, and differentially expressed mRNAs. MSTRG.25116.1 was identified as a potential regulator of chicken abdominal preadipocyte adipogenic differentiation by acting as a transcriptional trans-regulator of fatty acid amide hydrolase (FAAH) gene expression and/or a ceRNA that post-transcriptionally mediates FAAH gene expression by sponging gga-miR-1635.

Keywords: adipogenic differentiation; chicken; competing endogenous RNA; lncRNAs.

Figure 1

Identification of DE-lncRNAs during abdominal adipogenic differentiation in chickens. (A) Heatmap of DE-lncRNAs in chicken abdominal adipocytes from five differentiation stages; (B) volcano maps of the DE-lncRNAs in the four comparison groups (A0 vs. A12, A12 vs. A48, A48 vs. A72, and A72 vs. A120); (C) Venn analysis of the DE-lncRNAs from the four comparison groups.

Figure 2

qRT-PCR validation of lncRNAs’ expression. (A) Detection of lncRNAs’ expression in abdominal adipocytes from eight differentiation stages using qRT-PCR. All lncRNA expression levels are shown as fold change versus that of the 0 h group; (B) Comparative analysis of lncRNA expression from qRT-PCR and RNA-seq data. The x-axis indicates the lncRNAs tested and the y-axis indicates the log₂ foldchange. Red represents the expression data from RNA-seq and blue represents that from qRT-PCR. * p < 0.05 and ** p < 0.01.

Figure 3

KEGG enrichment analysis of target genes of DE-lncRNAs. Significantly enriched signaling pathways of cis-target genes (A) and trans-target genes (B) of DE-lncRNAs in the four comparison groups.

Figure 4

Short time-series expression analysis of DE-lncRNAs during abdominal adipogenic differentiation. (A) The expression profiles of DE-lncRNAs in cluster 1 containing profile 40 and profile 42; (B) the expression profiles of DE-lncRNAs in cluster 2 containing profile 1; (C) the expression profiles of DE-lncRNAs in cluster 3 containing profile 8. The x-axis indicates differentiation stages including 0 h (A0), 12 h (A12), 48 h (A48), 72 h (A72), and 120 h (A120). The y-axis indicates the log₂ foldchange in their expression. The colored lines represent the expression pattern of lncRNAs.

Figure 5

WGCNA of all expressed lncRNAs in chicken abdominal preadipocytes and differentiated adipocytes. (A) Hierarchical clustering dendrograms showing 35 modules of co-expressed lncRNAs. Color bar indicates the modules of co-expressed lncRNAs; (B) visualization of the eigengene network representing the relationships among the modules; (C) module-trait association heatmap where each row corresponds to a module eigengene and the columns correspond to a trait. Each cell contains the correlation and p value for the corresponding differentiation stages. The scale bar indicates the color coding for the correlations, with blue to red indicating low to high correlations, respectively.

Figure 6

Visualization of GS vs. MM and lncRNAs’ expression levels in seven modules. (A) A scatterplot of GS for differentiation stage vs. MM in seven modules. The cor means the correlation between GS and MM. The p value represents the significance of correlation between GS and MM; (B) Heatmap and bar-plot representing the expression levels of lncRNAs in 15 samples (from left to right: A0-1, A0-2, A0-3, A12-1, A12-2, A12-3, A48-1, A48-2, A48-3, A72-1, A72-2, A72-3, A120-1, A120-2, and A120-3) in seven modules. The color ranging from green to red in the heatmap indicates low to high expression levels.

Figure 7

The lncRNA–miRNA–mRNA ceRNA networks mediated by key lncRNAs. (A) The ceRNA networks of key lncRNAs, DE-miRNAs, and DE-mRNAs; (B,C) RNA-seq expression analysis of gga-miR-1635 and FAAH gene in chicken abdominal preadipocytes from different differentiation stages; (D) correlation analysis of the expression levels of MSTRG.25116.1, gga-miR-1635, and FAAH gene from RNA-seq during adipogenic differentiation in chicken abdominal preadipocytes. The red dots indicate the expression levels of genes that were represented on y-axis.