Chi-Circ_0006511 Positively Regulates the Differentiation of Goat Intramuscular Adipocytes via Novel-miR-87/CD36 Axis

Figure 1. Basic information of chi-circ_0006511. (A) Clustering heatmap of differentially expressed circRNAs between GIMPA and GIMA; (B)chi_circ_0006511 sequence information; (C) Electrophoresis of PCR products of Divergent primer and Convergent primer in Rnase R (+) and Rnase R (−) samples, “” indicates Divergent primer, “” indicates Convergent primer.

Figure 2. Chi-circ_0006511 overexpression promotes GIMPA differentiation. (A) Temporal expression of chi-circ_0006511 during GIMPA differentiation, capital letters indicated extremely significant difference (p < 0.01), lowercase letters indicated significant difference (p < 0.05); (B) Green fluorescent protein (GFP) characterizes the infection efficiency of vectors; (C) the overexpression efficiency of chi-circ_0006511. (D) The Bodipy and Oil red O staining reveals the lipid droplets in cells, Bodipy fluorescence was quantified with ImageJ; (E) the mRNA expression level of adipocytes differentiation related genes. Three biological replicates were set up for each group. Data are expressed as means ± SD. “**” indicated extremely significant difference (p < 0.01), “*” indicated significant difference (p < 0.05).

Figure 3. Chi-circ_0006511 knockdown inhibits GIMPA differentiation. (A) Efficiency detection of three siRNA knockdown of chi-circ_0006511; (B) The Bodipy and Oil red O staining reveals the lipid droplets in cells; (C) the mRNA expression level of adipocytes differentiation related marker genes. Data are expressed as means ± SD. “**” indicated extremely significant difference (p < 0.01), “*” indicated significant difference (p < 0.05).

Figure 4. Subcellular localization of chi-circ_0006511 and identification of its targeting relationship with novel-miR-87 as a ceRNA. (A) The nucleocytoplasmic localization of chi-circ_0006511 was detected by qPCR, U6 was used as a nuclear reference, and GAPDH was used as a cytoplasmic control to correct the expression level of chi-circ_0006511. (B) Subcellular localization detection of chi-circ_0006511 by FISH; (C) Venn diagram of chi-circ_0006511 as a ceRNA-bound miRNA prediction by MiRanda, RNAHybrid and coexpression analysis of RNA-seq(Demir-down); (D) The Minimum free energy prediction of the intersection of 10 miRNAs binding to chi-circ_0006511 by RNAHyrid; (E) novel-miR-87 was down-regulated by chi-circ_0006511 overexpression; (F) novel-miR-87 was up-regulated by chi-circ_0006511 knockdown; (G) qPCR detected the expression of novel-miR-87 with chi-circ_0006511 pull down assay; (H) Dual-luciferase reporter assay to verify the targeting relationship between novel-miR-87 and chi-circ_0006511. Data are expressed as means ± SD. “**” indicated extremely significant difference (p < 0.01).

Figure 5. The effect of novel-miR-87 on GIMPA differentiation. Efficiency of novel-miR-87 mimics (A) and inhibitor (D); The Bodipy and Oil red O staining reveals the lipid droplets novel-miR-87 mimics (B) and inhibitor (E); (C,F) the mRNA expression level of adipocytes differentiation related marker genes. Data are expressed as means ± SD. “**” indicated extremely significant difference (p < 0.01), “*” indicated significant difference (p < 0.05).

Figure 6. Novel-miR-87 target gene prediction and targeting identification. (A) Venn diagram of RNAHybrid and RNA-seq prediction of novel-miR-87 target genes; (B) KEGG analysis of intersecting target genes, showed the gene interaction map enriched with adipocyte differentiation-related pathways by Cytoscape; (C) CD36 mRNA expression level was up-regulated with overexpression of chi-circ_0006511. (D) CD36 mRNA expression level was down-regulated with knockdown of chi-circ_0006511; (E) novel-miR-87 mimics down-regulated CD36 mRNA expression; (F) novel-miR-87 inhibitor down-regulated CD36 mRNA expression; (G) The dual-luciferase reporter gene assay reflected the targeting relationship between novel-miR-87 and CD36. Data are expressed as means ± SD. “**” indicated extremely significant difference (p < 0.01).

Figure 7. Effects of CD36 on GIMPA differentiation. (A) CD36 sgRNA design and monoclonal verification; (B) CD36 expression level verification by qPCR and western blot; (C) The Bodipy and Oil red O staining reveals the accumulation of the lipid droplets; (D) the mRNA expression level of adipocytes differentiation related marker genes. Data are expressed as means ± SD. “**” indicated extremely significant difference (p < 0.01), “*” indicated significant difference (p < 0.05).

Figure 8. The model of chi-circ_0006511 regulating goat intramuscular adipocyte differentiation. “→” indicate promotion, “⟞” indicates inhibition, and “⇢” indicate that it is in speculation.