CircRNA Profiling Identifies Candidates Associated with Abdominal Fat Deposition in Ducks

Figures

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  Figure 3. Functional GO annotation and KEGG pathway enrichment analysis of host genes of differentially expressed circular RNAs. (A) GO enrichment pie chart showing the 20 most enriched terms across the three major categories: biological processes, cellular components, and molecular functions. The concentric circles, from the outer to the inner, represent, respectively, the coordinate scale for gene counts (color-coded by category), background gene counts and statistical significance (p-value), counts of specific differentially expressed circRNA host genes, and Rich factor values plotted against a background grid (0.1 per square). (B) A bubble plot showing selected lipid-related GO terms enriched in DE circRNA host genes. (C) KEGG enrichment pie chart showing the top 20 enriched pathways. (D) A bubble plot showing selected lipid metabolism pathways enriched by differentially expressed circRNA host genes.

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  Figure 4. RT-qPCR validation of selected DEcircRNAs and their corresponding host genes in Peking duck abdominal adipose tissue (n = 8 per group). (A) Relative expression levels of seven selected DEcircRNAs in the HF and LF groups. (B) Relative expression levels of host genes corresponding to 7 selected DEcircRNAs. The mean expression level in the LF group was normalized to 1. * p-value < 0.05, ** p-value < 0.01.

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  Figure 1. Alignment characteristics and genomic features of circRNA sequencing reads from abdominal adipose tissue of Peking ducks. (A) Distribution of mapped reads in exons, introns, and intergenic regions in the HF and LF groups. (B) Histogram of the length distribution of identified circRNAs. (C) Distribution of circRNA expression abundance based on the number of reads supported by back-splicing junctions (BSJ). (D) Chromosomal distribution map of identified circRNAs. (E) Distribution map of circRNA types.

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  Figure 2. Differential expression profiles and clustering analysis of circRNAs between the HF and LF groups. (A) A Venn diagram showing unique and overlapping circRNA candidates identified in abdominal adipose tissue from the HF and LF groups. (B) A hierarchical clustering heatmap used to assess the overall expression patterns of 212 significantly DE circRNAs across biological replicates. (C) A statistical bar chart summarizing the total number of significantly upregulated and downregulated DE circRNAs. (D) A volcano plot showing the overall transcriptional variation and statistical significance of all identified transcripts. Data points are displayed using a unique blue-yellow-red gradient, corresponding to significantly downregulated (blue), statistically insignificant (yellow), and significantly upregulated (red) circRNAs, respectively. (E) A radar chart illustrating the multidimensional characteristics of the top 20 differentially expressed circRNAs. These candidate circRNAs were selected and ranked based on the thresholds of p-value < 0.05 and |log2 FC| ≥ 1, where the orange regions represent significantly upregulated transcripts and the blue regions represent significantly…

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  Figure 5. Mapping the ceRNA regulatory network between key circular RNAs and adipogenesis targets. (A) Statistical bar chart of the ceRNA network; (B) A Sankey diagram illustrating the directed flow and multi-database intersections among candidate circular RNAs (left nodes), miRNAs (middle nodes), and related target genes (right nodes) during abdominal fat metabolism; (C) Relative expression levels of candidate circRNAs in abdominal fat tissue from LF group and HF group (n = 8 per group, Mean ± S.E.M). ** p-value < 0.01.

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  Figure 6. Adipogenic differentiation of primary abdominal preadipocytes and expression patterns of candidate circRNAs. (A–F) Representative images of Oil Red O-stained primary abdominal preadipocytes cultured under control or adipogenic induction conditions. (A,C,E) Uninduced control cells cultured for 2, 4, and 6 days, respectively. (B,D,F) Adipogenesis-induced cells cultured for 2, 4, and 6 days, respectively. Images were acquired using a 20× objective. Scale bar = 50 μm. (G–I) Relative expression levels of novel_circ_003710, novel_circ_010207, novel_circ_013157, and novel_circ_011631 in adipogenesis-induced cells and their time-matched controls on days 2 (G), 4 (H), and 6 (I), as determined by RT-qPCR. ABF-Y2, ABF-Y4, and ABF-Y6 represent cells subjected to adipogenic induction for 2, 4, and 6 days, respectively; ABF-Z2, ABF-Z4, and ABF-Z6 represent the corresponding uninduced controls. Expression in each time-matched control group was normalized to 1. Data are presented as the mean ± SEM from five independent biological replicates (n = 5 per group). * p-value < 0.05 and ** p-value < 0.01.

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  Figure 7. Temporal expression dynamics of candidate DE circRNAs during the adipogenic differentiation of primary duck abdominal preadipocytes. (A) Overall temporal expression profiles of novel_circ_003710, novel_circ_010207, novel_circ_013157, and novel_circ_011631 before adipogenic induction (D0) and after 2, 4, and 6 days of induction (D2, D4, and D6, respectively). (B–E) Individual expression profiles of novel_circ_003710 (B), novel_circ_010207 (C), novel_circ_013157 (D), and novel_circ_011631 (E), as determined by RT-qPCR. Relative expression levels were normalized to the corresponding expression levels at D0. Data are presented as the mean ± SEM from five independent biological replicates (n = 5 per group, Mean ± S.E.M). Statistical differences between each post-induction time point and D0 were assessed using one-way ANOVA followed by Dunnett’s test. ** p-value < 0.01 and ns, not significant.