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

Abstract

Goats are an important livestock and goat meat is essential to local people. The intramuscular fat (IMF) content has a great influence on the quality of goat meat. The intramuscular preadipocytes differentiation is closely related to the IMF deposition; however, its potential regulatory mechanisms remain unclear. CircRNAs were revealed to be involved in multiple biological progressions. In this study, we took primary goat intramuscular preadipocyte (GIMPA) as the study model to verify the function and mechanism of chi-circ_0006511, which was abundant and up-regulated in mature adipocytes (GIMA). The results showed that the expression level of chi-circ_0006511 gradually increased in the early stage of GIMPA differentiation, and chi-circ_0006511 was confirmed to promote GIMPA lipid droplets aggregation and up-regulate the adipogenic differentiation determinants, further promoting GIMPA differentiation. Mechanistically, chi-circ_0006511 exerts its function by sponging novel-miR-87, thereby regulating the expression of CD36. The results from this study provided novel significant information to better understand the molecular regulatory mechanism of intramuscular preadipocytes differentiation, thereby providing a new reference for the intramuscular fat adipogenesis in goats.

Keywords: CD36; adipocyte differentiation; circRNA; goat; novel-miR-87.

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.