CircVPS13C Promotes Intramuscular Adipogenesis via MiR-5606-X-ECHDC3 Axis in Yaks (Bos grunniens)

Figure 1. Characterization and expression of circVPS13C. (A) Heatmap of differentially expressed circRNAs; (B) schematic diagram of circVPS13C circular formation; (C) schematic diagram of circVPS13C ring formation; (D) PCR analysis to detect the expression of circVPS13C in cDNA and gDNA; (E) comparison of circVPS13C and VPS13C mRNA abundance after RNase R digestion; (F) localization of circVPS13C in the nucleus and cytoplasm; (G) sankey diagram displaying the interactions between circRNA, miRNA, and mRNA; (H) KEGG enrichment of source genes of differentially expressed circRNAs. The RNase R assay and nuclear-cytoplasmic fractionation were performed in triplicate and repeated 3 times (n = 9). Values are presented as mean ± SEM.

Figure 2. Effects of circVPS13C on proliferation and differentiation of YIMAs. (A) Expression level of circVPS13C; (B) expression of differentiation marker genes; (C) BODIPY staining; (D) Oil Red O staining; (E) semi-quantification of Oil Red O staining; (F) expression of proliferation marker genes; (G) EdU staining; (H) quantitative analysis of EdU staining using ImageJ; (I) CCK-8 measurement. The CCK-8 assay was performed in duplicate and repeated three times (n = 6), and the other experiments were performed in triplicate and repeated three times (n = 9). Values are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns = not significant.

Figure 3. Effects of circVPS13C siRNA on proliferation and differentiation of YIMAs; (A) interference efficiency of circVPS13C; (B) expression of differentiation marker genes; (C) BODIPY staining; (D) Oil Red O staining; (E) semi-quantification of Oil Red O staining; (F) expression of proliferation marker genes; (G) EdU staining; (H) quantitative analysis of EdU staining using ImageJ; (I) CCK-8 measurement. The CCK-8 assay was performed in duplicate and repeated three times (n = 6), and the other experiments were performed in triplicate and repeated three times (n = 9). Values are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns = not significant.

Figure 4. CircVPS13C regulates yak fat deposition via the circVPS13C/miR-5606-x/ECHDC3 axis. (A) Schematic diagram of dual-luciferase reporter gene vector constructs for circVPS13C and ECHDC3; (B) luciferase activity of YIMAs at 48 h after transfection with WT or MUT circVPS13C and miR-5606-x mimics; (C) expression level of circVPS13C after miR-5606-x inhibition; (D) the expression of PPARγ after co-transfection with circVPS13C siRNA and miR-5606-x inhibitor; (E) luciferase activity of YIMAs at 48 h after transfection with WT or MUT ECHDC3 and miR-5606-x. The dual-luciferase reporter assay and co-transfection assay were performed in triplicate and repeated three times (n = 9). Values are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns = not significant.

Figure 5. Effects of miR-5606-x on proliferation and differentiation of YIMAs. (A) Overexpression efficiency of miR-5606-x; (B) differentiation marker gene expression after miR-5606-x mimics transfection; (C) transfection of miR-5606-x mimic followed by BODIPY staining; (D) Oil Red O staining after transfection with mimics; (E) semi-quantification of Oil Red O staining after transfection with mimics; (F) CCK-8 detection after transfection of miR-5606-x mimic; (G) EdU staining after transfection of miR-5606-x mimic; (H) quantitative analysis of EdU staining using ImageJ after transfection with mimics; (I) expression of proliferation marker genes after transfection with miR-5606-x mimics; (J) interference efficiency of miR-5606-x; (K) expression of differentiation marker genes after transfection with inhibitor; (L) Oil Red O staining after transfection with inhibitor; (M) BODIPY staining after transfection with inhibitor; (N) semi-quantification of Oil Red O staining after transfection with inhibitor; (O) CCK-8 assay after transfection with inhibitor; (P) EdU staining after transfection with inhibitor; (Q) quantitative analysis of EdU staining using ImageJ after transfection with inhibitor;…

Figure 6. Effects of ECHDC3 on proliferation and differentiation of YIMA. (A) Overexpression efficiency of ECHDC3 in yak intramuscular preadipocytes; (B) knockdown efficiency of ECHDC3 siRNA; (C) effects of ECHDC3 overexpression on adipogenic marker gene expression; (D) Oil Red O staining after ECHDC3 overexpression; (E) BODIPY staining after overexpressing ECHDC3; (F) impact of ECHDC3 overexpression on proliferation-related gene expression; (G) EdU incorporation assay evaluating proliferative activity upon ECHDC3 overexpression; (H) cell proliferation assessed by CCK-8 assay after ECHDC3 overexpression; (I) influence of ECHDC3 knockdown on adipogenic gene expression; (J) BODIPY staining after ECHDC3 interference; (K) Oil Red O staining after ECHDC3 interference; (L) effects of ECHDC3 interference on proliferation-related gene expression; (M) EdU staining assessing cell proliferation after ECHDC3 knockdown; (N) CCK-8 assay measuring cell proliferation after ECHDC3 knockdown. The CCK-8 assay was performed in duplicate and repeated 3 times (n = 6), and the other experiments were performed in triplicate and repeated three times (n = 9). Values are presented as mean ± SEM; ** p < 0.01, *** p <…

Figure 7. A model for circVPS13C regulation of preadipocyte deposition in yaks.