Circ_0011446 Regulates Intramuscular Adipocyte Differentiation in Goats via the miR-27a-5p/FAM49B Axis

Abstract

Intramuscular fat (IMF), or marbling, is a critical indicator of goat meat quality. Non-coding RNAs play a key role in the formation and deposition of IMF in vertebrates by regulating genes involved in its synthesis, degradation, and transport. The competing endogenous RNA (ceRNA) hypothesis identifies circular RNAs (circRNAs) as natural "sponges" for microRNAs (miRNAs). However, the precise mechanisms of circRNAs in goat IMF remain poorly understood. In the current study, we utilized existing sequencing data to construct a ceRNA regulatory network associated with intramuscular adipogenesis and fat deposition in goats. Our goal was to elucidate the post-transcriptional regulatory mechanism of family with sequence similarity 49 member B (FAM49B). Functionally, FAM49B was found to inhibit the differentiation of intramuscular preadipocytes and to directly interact with miR-27a-5p. Mechanistically, dual-luciferase reporter assays and quantitative real-time PCR (qRT-PCR) confirmed the interaction between circ0011446 and miR-27a-5p. Circ0011446 enhanced the expression of FAM49B mRNA and protein through post-transcriptional regulation. As a ceRNA, circ0011446 competitively binds miR-27a-5p, preventing miR-27a-5p from degrading FAM49B. In conclusion, our findings demonstrate that circ0011446 suppresses goat adipogenic differentiation of intramuscular preadipocytes by regulating the expression of the downstream target gene FAM49B through miR-27a-5p sequestration. This study provides a reference for goat meat quality or livestock breeding.

Keywords: FAM49B; circ_0011446; goat; intramuscular adipocyte differentiation; miR-27a-5p.

Figure 1

Identification of circ_0011446. (A). Cyclization diagram of circ_0011446 and sequence diagram of the back-splicing site analysis. (B). circ_0011446 ring-forming identification. M is the marker; RNase R (+) and RNase R (−) represent whether there is RNase R digestion; and represent the convergent prime and divergent prime, respectively. (C). Relative circ_0011446 expression during goat intramuscular adipocyte differentiation. Different lowercase letters indicate significant differences between groups according to one-way ANOVA (p < 0.01).

Figure 2

circ_0011446 inhibits adipogenic differentiation in goat. (A). Overexpression efficiency of circ_0011446. (B,F). Images of mature intramuscular adipocytes stained with Oil Red O (left panel) and OD value determination (right panel) following circ_0011446 overexpression (B) and knockdown (F), respectively. Oil Red O staining signal was quantified by absorbance at 490 nm. (C,G). Staining of mature intramuscular adipocytes with Bodipy (left panel) and fluorescence area quantification (right panel) after overexpression (C) and knockdown (G) of circ_0011446, respectively. (D,H). mRNA expression levels of adipogenic marker genes following circ_0011446 overexpression (D) and knockdown (H), respectively. (E). Interference efficiency of circ_0011446. All data are presented as mean ± SEM, * p < 0.05, ** p < 0.01.

Figure 3

circ_0011446 acts as a miR-27a-5p sponge. (A). Nucleocytoplasmic separation demonstrated the predominant cytoplasmic localization of circ_0011446. (B). Subcellular localization of circ_0011446 shown by FISH. (C). Potential miRNA binding sites of circ_0009659 predicted by RNAHybrid. (D,E). Expression changes of miR-27a-5p after overexpression (D) or knockdown (E) of circ_0011446. (F). Dual-luciferase reporter assay results showed that miR-27a-5p targets circ_0011446. All data are presented as mean ± SEM, ** p < 0.01.

Figure 4

Effect of miR-27a-5p on differentiation of goat intramuscular preadipocytes. (A,E). Overexpression (A) or knockdown (E) efficiency of miR-27a-5p detected by qRT-PCR. (B,F). The effect of miR-27a-5p overexpression (B) or knockdown (F) on adipogenic differentiation was quantitatively assessed by Oil Red O staining, with the OD value of Oil Red O dye at 490 nm measured. (C,G). Bodipy staining analysis of miR-27a-5p overexpression (C) or knockdown (G) in intramuscular preadipocytes. (D,H). The effect of miR-27a-5p overexpression (D) or knockdown (H) on adipogenic marker gene expression was detected by qRT-PCR. * p < 0.05, ** p < 0.01, compared to that of NC.

Figure 5

Prediction and validation of the targeting relationship between miR-27a-5p and FAM49B. (A). TargetScan, MIRDB, and Starbase predicted miR-27a-5p targeting FAM49B. (B,C). The effect of miR-27a-5p overexpression (B) and knockdown (C) on FAM49B expression was detected by qRT-PCR. (D). A dual-luciferase reporter assay verified the targeting relationship between miR-27a-5p and FAM49B (Right panel), with construction of wild-type and mutant vectors (Left panel). The data were shown as the mean ± SEM (n = 3) (** p < 0.01).

Figure 6

Role of FAM49B in goat intramuscular preadipocyte differentiation. (A,E). FAM49B overexpression (A) or knockdown (E) efficiency. (B,F). Oil Red O staining of mature adipocytes (left) and OD value at 490 nm (right) after FAM49B overexpression (B) or knockdown (F). (C,G). Bodipy staining of mature adipocytes (left) and fluorescence area quantification (right) after FAM49B overexpression (C) or knockdown (G). (D,H). mRNA levels of adipogenic markers after FAM49B overexpression (D) or knockdown (H). Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01.

Figure 7

Model of circ_0011446 functioning as a ceRNA to regulate FAM49B expression by sponging miR-27a-5p, thus inhibiting the differentiation of goat intramuscular adipocyte.