circARID1A Inhibits Tail Fat Cell Differentiation in Guangling Large-Tailed Sheep by Regulating the miR-493-3p/YTHDF2 Axis

Abstract

The Guangling Large-Tailed sheep is renowned for its unique tail fat deposition, with a significant proportion of its total body fat being localized in the tail region. Fat deposition is a complex biological process regulated by various molecular mechanisms. Our previous studies have identified a large number of differentially expressed circular RNAs (circRNAs) in the tail adipose tissue of the Guangling Large-Tailed sheep. These circRNAs may play a pivotal role in the process of fat deposition. Given the potential regulatory functions of circRNAs in adipose metabolism, investigating their roles in tail fat deposition is of significant scientific importance. In this study, we identified novel circARID1A. Using various experimental methods, including lentivirus infection, RNase R treatment, actinomycin D assay, qPCR, western blotting, and dual-luciferase reporter assays, we determined that circARID1A inhibits the expression of miR-493-3p through competitive binding, thereby regulating adipocyte differentiation. Further research revealed that miR-493-3p promotes adipocyte differentiation by targeting YTH domain family 2 (YTHDF2), and this regulatory effect is also influenced by circARID1A. In conclusion, our findings suggest that circARID1A inhibits tail fat cell differentiation in the Guangling Large-Tailed sheep through the circARID1A/miR-493-3p/YTHDF2 axis, providing theoretical support for improving meat quality and fat deposition in sheep.

Keywords: Guangling Large-Tailed sheep; YTHDF2; adipocyte differentiation; circARID1A; miR-493-3p.

Figure 1

Characterization and identification of circARID1A. (A) circARID1A is generated from exons 2 and 4 of the ARID1A gene. (B) Sanger sequencing confirmed the presence of circARID1A. (C) PCR analysis using divergent and convergent primers on cDNA and gDNA. (D) Relative expression levels of circARID1A and ARID1A after RNase R treatment. (E) Relative expression levels of circARID1A and ARID1A after ActD treatment. (F) Relative expression levels of circARID1A in the nucleus and cytoplasm after nuclear–cytoplasmic separation. ***: p < 0.001.

Figure 2

The effect of circARID1A on adipocyte differentiation. (A) Expression levels of circARID1A and ARID1A mRNA in adipocytes after transfection with circARID1A overexpression vector and control vector NC. (B) Expression levels of circARID1A and ARID1A mRNA after transfection with shRNA and sh-NC. (C) mRNA expression levels of four adipogenic marker genes after circARID1A overexpression and knockdown. (D,E) Protein expression levels of four adipogenic marker genes after circARID1A overexpression and knockdown. (F) Oil Red O staining results of adipocytes differentiated for 6 days. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 3

circARID1A acts as a molecular sponge for miR-493-3p. (A) Schematic diagrams of circARID1A-wt and circARID1A-mut luciferase reporters. (B) Luciferase activity in 293T cells co-transfected with circARID1A-wt or circARID1A-mut plasmids and miR-493-3p mimic or miR-493-3p NC for 48 h. (C,D) RIP assays in adipocytes using anti-AGO2 antibody or IgG, with qPCR measuring enrichment levels of circARID1A and miR-493-3p. (E) Expression levels of miR-493-3p after overexpression or knockdown of circARID1A. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 4

Effects of miR-493-3p on adipocyte differentiation. (A) Overexpression and inhibition of miR-493-3p. (B) mRNA expression levels of four adipocyte differentiation marker genes following overexpression and inhibition of miR-493-3p. (C,D) Protein expression levels of the four adipocyte differentiation marker genes after miR-493-3p overexpression and inhibition. (E) Oil Red O staining results of adipocytes after 6 days of differentiation. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 5

Target relationship between YTHDF2 and miR-493-3p, and regulation by circARID1A. (A) Schematic diagrams of YTHDF2 3′UTR WT and YTHDF2 3′UTR MUT luciferase reporters. (B) Luciferase activity measured 48 h after co-transfection of wild-type and mutant luciferase reporters with miR-493-3p mimic or miR-493-3p NC into 293T cells. (C) mRNA expression levels of YTHDF2 after overexpression or inhibition of circARID1A. (D,E) Protein expression levels of YTHDF2 after overexpression or inhibition of miR-493-3p. (F,G) Protein expression levels of YTHDF2 after overexpression or inhibition of circARID1A. *: p < 0.05, **: p < 0.01.

Figure 6

Effects of YTHDF2 on adipocyte differentiation. (A) Overexpression and knockdown of YTHDF2. (B) mRNA expression levels of four adipocyte differentiation marker genes following YTHDF2 overexpression and knockdown. (C,D): Protein expression levels of the four adipocyte differentiation marker genes after YTHDF2 overexpression and knockdown. (E) Oil Red O staining results of adipocytes after 6 days of differentiation. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 7

miR-493-3p Alleviates the regulation of YTHDF2 by circARID1A. (A) mRNA expression levels of YTHDF2 and adipocyte differentiation marker genes in the rescue experiment. (B,C) Protein expression levels of YTHDF2 in the rescue experiment. (D) Oil Red O staining results of adipocyte differentiation after 6 days in the rescue experiment. *: p < 0.05, **: p < 0.01.