MiR-218-5p Affects Subcutaneous Adipogenesis by Targeting ACSL1, a Novel Candidate for Pig Fat Deposition

Abstract

As a centre enzyme in fatty acid activation, acyl-CoA synthetase long-chain family member 1 (ACSL1) plays an important role in body lipid homeostasis. However, the functions of ACSL1 in the subcutaneous adipogenesis of pigs are largely unknown. In the present study, we found that the expression of ACSL1 significantly increased during the process of porcine preadipocyte differentiation. Moreover, silencing of ACSL1 in preadipocytes decreased levels of triglyceride and adipogenic-related markers, including FABP4, APOE, and FASN (p < 0.01), and simultaneously increased levels of lipolytic-related markers, such as ATGL and HSL (p < 0.05). Conversely, overexpression of ACSL1 in preadipocytes increased levels of triglyceride and FABP4, APOE, and FASN (p < 0.01), and reduced levels of ATGL and HSL (p < 0.05). Luciferase reporter assays revealed that ACSL1 is a target of miR-218-5p, which can reduce the mRNA and protein levels of ACSL1 by directly binding the 3' untranslated region of ACSL1. Furthermore, miR-218-5p has an inhibition role in porcine preadipocyte differentiation by suppressing ACSL1 expression. Taken together, these data provide insights into the mechanism of the miR-218-5p/ACSL1 axis in regulating subcutaneous fat deposition of pigs.

Keywords: ACSL1; miR-218-5p; miRNAs; pigs; subcutaneous fat deposition.

Figure 1

Differentiation of porcine preadipocytes and the expression pattern of ACSL1 in differentiated preadipocytes. Morphological (A) and triglyceride content (B) changes of preadipocytes at d 0, d 2, d 4, and d 8. (C): mRNA levels of ACSL1 at different differentiation stages. D 0, d 2, d 4, and d 8: 0 h, 48 h, 96 h, and 192 h after adipogenic induction. Data are shown as mean ± SEM. n = 3 per group. **: p < 0.01.

Figure 2

Effect of siRNA interference with ACSL1 on preadipocyte differentiation. Interfering with ACSL1 inhibits preadipocyte differentiation phenotype (A) and triglyceride content (B) at d 8. (C) Effect of interference with ACSL1 on preadipocyte differentiation-related genes at d 2. D 2 and d 8: 48 h and 192 h after adipogenic induction. Data are shown as mean ± SEM. n = 3 per group. *: p < 0.05, **: p < 0.01, ns: p < 0.05.

Figure 3

Effect of overexpression of ACSL1 on preadipocyte differentiation. Effect of overexpression of ACSL1 on preadipocyte differentiation phenotype (A) and triglyceride content (B) at d 8. (C) Effect of overexpression of ACSL1 on preadipocyte-differentiation-related genes at d 2. D 2 and d 8: 48 h and 192 h after adipogenic induction. Data are shown as mean ± SEM. n = 3 per group. *: p < 0.05, **: p < 0.01, ns: p < 0.05.

Figure 4

MiR-218-5p targets the 3′UTR of ACSL1. (A) The conserved mature sequence of miR-218-5p in different species. (B) Conservative analysis of the binding site of miR-218-5p to the 3′UTR of ACSL1 in different species. (C) The wild-type (wt) and mutant (mut) 3′UTR fluorescent reporter vector plasmids of ACSL1 were aligned with miR-218-5p. (D) Luciferase activity of pmirGLO-ACSL1 3′UTR-wt and pmirGLO-ACSL1 3′UTR-mut co-transfected with miR-218-5p mimics for 24 h. Data in D are represented as mean ± SEM of six experimental replicates. **: p < 0.01, ns: p < 0.05.

Figure 5

MiR-218-5p inhibits preadipocyte differentiation. (A) MiR-218-5p overexpression in preadipocytes at d 2. (B) Effect of miR-218-5p overexpression and inhibitor on preadipocyte differentiation at d 8. (C) Effect of miR-218-5p overexpression and inhibitor on triglyceride content at d 8. (D) Effect of miR-218-5p overexpression on ACSL1 at mRNA and protein levels at d 2. (E) Effect of miR-218-5p inhibitor on ACSL1 at mRNA and protein levels at d 2. Treatment group: adipocytes treated with miR-218-5p mimics (left) or inhibitor (right). D 2 and d 8: 48 h and 192 h after adipogenic induction. Data are shown as mean ± SEM. n = 3 per group, *: p < 0.05, **: p < 0.01.

Figure 6

MiR-218-5p targeting ACSL1 affects preadipocyte differentiation. Effect of siRNA-ACSL1, miR-218-5p inhibitor, and co-transfection of both on preadipocyte differentiation at d 8 was determined by Oil Red O staining (A) and triglyceride content detection (B). D 8: 192 h after adipogenic induction. “+” and “−”: the materials (siRNA-NC, miR-218-5p inhibitor-NC, siRNA-ACSL1, and miR-218-5p inhibitor) were transfected (+) or not transfected (−) into preadipocytes. Data are shown as mean ± SEM. n = 3 per group, *: p < 0.05, **: p < 0.01, ns: p < 0.05.