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. 2023 Feb 9;24(4):3501.
doi: 10.3390/ijms24043501.

circINSR Inhibits Adipogenic Differentiation of Adipose-Derived Stromal Vascular Fractions through the miR-152/ MEOX2 Axis in Sheep

Bishi Zhao  1 , Hanyue Zhang  1 , Dan Zhao  1 , Yu Liang  1 , Liying Qiao  1 , Jianhua Liu  1 , Yangyang Pan  1 , Kaijie Yang  1 , Wenzhong Liu  1
Affiliations

circINSR Inhibits Adipogenic Differentiation of Adipose-Derived Stromal Vascular Fractions through the miR-152/ MEOX2 Axis in Sheep

Bishi Zhao et al. Int J Mol Sci. .

Abstract

Adipose tissue plays a crucial role in energy metabolism. Several studies have shown that circular RNA (circRNA) is involved in the regulation of fat development and lipid metabolism. However, little is known about their involvement in the adipogenic differentiation of ovine stromal vascular fractions (SVFs). Here, based on previous sequencing data and bioinformatics analysis, a novel circINSR was identified in sheep, which acts as a sponge to promote miR-152 in inhibiting the adipogenic differentiation of ovine SVFs. The interactions between circINSR and miR-152 were examined using bioinformatics, luciferase assays, and RNA immunoprecipitation. Of note, we found that circINSR was involved in adipogenic differentiation via the miR-152/mesenchyme homeobox 2 (MEOX2) pathway. MEOX2 inhibited adipogenic differentiation of ovine SVFs and miR-152 inhibited the expression of MEOX2. In other words, circINSR directly isolates miR-152 in the cytoplasm and inhibits its ability to promote adipogenic differentiation of ovine SVFs. In summary, this study revealed the role of circINSR in the adipogenic differentiation of ovine SVFs and its regulatory mechanisms, providing a reference for further interpretation of the development of ovine fat and its regulatory mechanisms.

Keywords: MEOX2; adipogenesis; circINSR; miR-152; sheep; stromal vascular fraction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of circINSR in ovine SVFs. (A) circINSR generated from the second exon of the INSR gene. (B) The existence of circINSR was verified via Sanger sequencing. (C) PCR analysis of divergent and convergent primers in cDNA and gDNA. (D) The relative expression of circINSR and INSR after RNase R treatment. (E) The relative expression of circINSR and INSR in ovine SVFs treated with ActD at the indicated time point. (F) Levels of circINSR in the nuclear and cytoplasmic fractions of ovine SVFs. ***: p < 0.001.
Figure 2
Figure 2
Effects of circINSR on adipogenic differentiation of ovine SVFs. (A) Two siRNAs targeting the circINSR junction site were constructed. (B) The relative expression of circINSR and INSR mRNA in ovine SVFs after transfection with the circINSR overexpression vector (pCD2.1-circINSR) or the control vector (pCD2.1-NC). (C) The relative expression of circINSR and INSR mRNA in ovine SVFs after transfection with two siRNAs or the control siRNA (si-NC). (D,E) The mRNA expression of 4 lipogenic genes in ovine SVFs that overexpress or interfere with circINSR. (F,G) The protein expression of 4 lipogenic genes in ovine SVFs that overexpress or interfere with circINSR. (H) Ovine SVFs were stained with ORO on day 10 after induction of differentiation. Scale bars: 100 μm. *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Figure 3
Figure 3
circINSR acts as a sponge for miR-152. (A) Schematic illustration of the circINSR-wt, circINSR-mut1, and circINSR-mut2 luciferase vectors. (B) The relative luciferase activities were detected in 293 T cells after transfection with circINSR-wt, circINSR-mut1, or circINSR-mut2 and miR152 mimics or mimics NC, respectively. (C,D) A RIP assay was carried out with anti-Ago2 antibodies or IgG in ovine SVFs after transfection with the miR-152 mimics or mimics NC, and qRT-PCR was then performed to detect the enrichment of circINSR and miR-152. (E) The relative expression of miR-152 in ovine SVFs that overexpress or interfere with circINSR. *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Figure 4
Figure 4
Effects of miR-152 on adipogenic differentiation of ovine SVFs. (A) Relative expression of miR-152 after transfection with miR-152 mimics or mimics NC and miR-152 inhibitors or inhibitors NC. (B,C) The mRNA expression of 4 lipogenic genes in ovine SVFs that overexpress or inhibit miR-152. (D,E) The protein expression of 4 lipogenic genes in ovine SVFs that overexpress or inhibit miR-152. (F) Ovine SVFs were stained with ORO on day 10 after induction of differentiation. Scale bars: 100 μm. *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Figure 5
Figure 5
MEOX2 is directly targeted by miR-152 and indirectly regulated by circINSR. (A) The relative mRNA levels of 4 candidate target genes in ovine SVFs that transfect with miR-152 inhibitors or inhibitors NC. (B) Schematic illustration of the MEOX2 3′-UTR-wt and MEOX2 3′-UTR-mut luciferase vectors. (C) The relative luciferase activities were detected in 293 T cells after transfection with MEOX2 3′-UTR-wt or MEOX2 3′-UTR-mut and miR152 mimics or mimics NC, respectively. (D,E) The protein expression of MEOX2 in ovine SVFs that overexpress or inhibit miR-152. (F,G) The protein expression of MEOX2 in ovine SVFs that overexpress or interfere with circINSR. (H) The mRNA expression of MEOX2 in ovine SVFs that overexpress or interfere with circINSR. *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Figure 6
Figure 6
Effects of MEOX2 on adipogenic differentiation of ovine SVFs. (A) Transfection efficiency of overexpression (PHBLV-MEOX2) or interference (shMEOX2-1 and shMEOX2-2) with MEOX2 based on the expression of GFP (green fluorescent protein) in ovine SVFs observed by fluorescence microscopy. Scale bars: 200 μm. (B) Relative expression of MEOX2 after transfection with PHBLV-MEOX2 or PHBLV-NC and shMEOX2-1, shMEOX2-2, or sh-NC. (C,D) The mRNA expression of 4 lipogenic genes in ovine SVFs that overexpress or interfere with MEOX2. (E,F) The protein expression of MEOX2 and 4 lipogenic genes in ovine SVFs that overexpress or interfere with MEOX2. (G) Ovine SVFs were stained with ORO on day 10 after induction of differentiation. Scale bars: 500 μm. *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Figure 7
Figure 7
circINSR upregulates the level of MEOX2 through miR-152. (A) The relative expression of circINSR and miR-152 after co-transfection of pCD2.1-circINSR and miR-152 mimics in ovine SVFs. (B) The relative expression of circINSR and miR-152 after co-transfection of si-circINSR and miR-152 inhibitors in ovine SVFs. (C,D) The mRNA expression of MEOX2 and 4 lipogenic genes in ovine SVFs that co-transfect with pCD2.1-circINSR and miR-152 mimics or si-circINSR and miR-152 inhibitors. (E,F) The protein expression of MEOX2 and 4 lipogenic genes in ovine SVFs that co-transfect with pCD2.1-circINSR and miR-152 mimics or si-circINSR and miR-152 inhibitors. (G) Ovine SVFs were stained with ORO on day 10 after induction of differentiation. Scale bars: 500 μm. *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Figure 8
Figure 8
Schematic representation of a model for the major molecular mechanisms of the “circINSR/miR-152/MEOX2” axis in ovine SVFs.
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