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. 2025 Apr;38(4):577-587.
doi: 10.5713/ab.24.0322. Epub 2024 Oct 25.

Chi-circ_0009659 modulates goat intramuscular adipocyte differentiation through miR-3431-5p/STEAP4 axis

Xin Li  1   2   3 , Hao Zhang  1   2   3 , Yong Wang  1   2   3 , Yanyan Li  1   2   3 , Youli Wang  1   2   3 , Yan Xiong  1   2   3 , Wei Liu  1   2   3 , Yaqiu Lin  1   2   3
Affiliations

Chi-circ_0009659 modulates goat intramuscular adipocyte differentiation through miR-3431-5p/STEAP4 axis

Xin Li et al. Anim Biosci. 2025 Apr.

Abstract

Objective: Circular RNAs (circRNAs) are widely involved in the regulation of lipid deposition in animals, but there are few reports on key circRNAs regulating intramuscular adipocyte differentiation in goats. Therefore, this study took an abundantly expressed in goat adipocytes chi-circ_0009659 as the object.

Methods: Based on the identification of back splicing site in chi-circ_0009659, its expression level during the goat intramuscular preadipocyte differentiation was detected by quantitative polymerase chain reaction (qPCR) . The chi-circ_0009659 loss-of-function and gain-of-function cell models were obtained by adenovirus and smarter silencer, respectively. and the adipocyte differentiation were explored by Oil Red O staining, Bodipy staining and qPCR. Its major cytoplasmic localization was determined by fluorescence in situ hybridization (FISH), nucleocytoplasmic separation and qPCR. The interaction between chi-circ_0009659, miR-3431-5p, and STEAP family member 4 (STEAP4) was verified by bioinformatics, RNA pull down and dual luciferase reporter assay.

Results: Silencing chi-circ_0009659 inhibited lipid droplet accumulation and the expression of differentiation-determining genes in goat intramuscular adipocytes, while overexpression of chi-circ_0009659 reversed these results. chi-circ_0009659 was predominantly localized to the cytoplasm and could regulate miR-3431 expression which in turn affects STEAP4. Consistent with expectations, miR-3431-5p acted as a negative regulator of GIMPA differentiation, while STEAP4 promoted differentiation.

Conclusion: We demonstrated chi-circ_0009659 positively regulates goat intramuscular preadipocyte differentiation by sponging miR-3431-5p to further regulate the expression of STEAP4. This research provides a new reference for in-depth understanding of the effects of circRNA on adipocyte differentiation.

Keywords: Adipocyte Differentiation; Circular RNA; Goat; STEAP Family Member 4.

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Conflict of interest statement

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

Figures

Figure 1
Figure 1
Identification of chi-circ_0009659. (A) Diagram of the source of circularization of chi-circ_0009659 and identification of the back-splicing site. (B) Electrophoresis of PCR products of Divergent primer and Convergent primer in Rnase R (+), Rnase R (−) and gDNA, “◂▶” indicated Divergent primer, “▶◂” indicated Convergent primer. (C) Expression levels of chi-circ_0009659 at different stages of goat intramuscular adipocyte differentiation. Groups that do not share the same uppercase letter are significantly (p<0.01) different from each other, and do not share the same lowercase letters are significantly (p<0.05) different from each other.
Figure 2
Figure 2
chi-circ_0009659 discourages differentiation of goat intramuscular adipocytes. (A) The efficiency of chi-circ_0009659 knockdown. (B), (F) Bodipy staining (green) and Oil Red O staining (red) revealed the lipids accumulation in GIMAs. (C) The mRNA relative expression of adipogenetic marker genes with chi-circ_0009659 knockdown. (D) Observation of the green fluorescent protein expression in GIMAs. (E) The efficiency of chi-circ_0009659 overexpression. (G) The mRNA relative expression of adipogenetic marker genes with chi-circ_0009659 overexpression. * p<0.05, ** p<0.01.
Figure 3
Figure 3
Prediction and identification of chi-circ_0009659 downstream miRNA. (A) Nucleocytoplasmic separation demonstrated the major cytoplasmic localization of chi-circ_0009659. (B) Subcellular localization of chi-circ_0009659 shown by FISH. (C) Chi-circ_0009659 potentially bound miRNAs predicted by RNAHybrid. (D) RNA pull down assay showed that miR-3431-5p was pulled down together with chi-circ_0009659. (E) miR-3431-5p decreased with overexpression of chi-circ_0009659. (F) miR-3431-5p was elevated with chi-circ_0009659 silencing. (G) Dual-luciferase reporter assay demonstrated miR-3431-5p targets chi-circ_0009659. * p<0.05, ** p<0.01.
Figure 4
Figure 4
miR-3431-5p is a negative regulator of goat intramuscular adipocyte differentiation. (A), (D) The efficiency of miR-3431-5p mimics/inhibitor. (B), (E) Bodipy staining (green) and Oil Red O staining (red) revealed the lipids accumulation in GIMAs. (C), (F) The mRNA relative expression of adipogenetic marker genes. * p<0.05, ** p<0.01.
Figure 5
Figure 5
STEAP4 promotes GIMAs differentiation as a downstream molecule of chi-circ_0009659 and miR-3431-5p. (A) The prediction of binding between miR-3431-5p and STEAP4 by RNAHybrid. (B) STEAP4 was upregulated by miR-3431-5p mimics and was down-regulated by miR-3431-5p inhibitor. (C) STEAP4 was upregulated by chi-circ_0009659 overexpression and was down-regulated by chi-circ_0009659 knockdown. (D) Dual-luciferase reporter confirmed that miR-3431-5p targets STEAP4. (E) Overexpression efficiency of STEAP in GIMAs. (F),(I) Bodipy staining (green) and Oil Red O staining (red) revealed the lipids accumulation in GIMAs. (G) The mRNA relative expression of adipogenetic marker genes with STEAP4 overexpression. (H) The efficiency of STEAP knockdown in GIMAs. (J) The mRNA relative expression of adipogenetic marker genes with STEAP4 knockdown. * p<0.05, ** p<0.01.
Figure 6
Figure 6
Schematic representation of chi-circ_0009659 promoting goat intramuscular adipocyte differentiation through miR-3431-5p/STEAP4 axis.
See this image and copyright information in PMC

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