Elsevier

Gene

Volume 666, 5 August 2018, Pages 158-164
Gene

Research paper
MiR-183 promotes preadipocyte differentiation by suppressing Smad4 in goats

https://doi.org/10.1016/j.gene.2018.05.022Get rights and content

Highlights

  • 1 miR-183 was up-regulated during goat preadipocyte differentiation.

  • 2 Overexpression miR-183 accelerated the differentiation of goat preadipocyte, while repressing miR-183 had opposite effects.

  • 3 Smad4 was validated as a target gene of miR-183 in goat and, decreasing Smad4 promoted goat adipogenesis.

  • 4 miR-183 promotes hircine preadipocytes differentiation through targeting Smad4.

Abstract

As a well-conserved microRNA, miR-183 is ubiquitously expressed in many tissues and cells including backfat and the 3T3-L1 adipocytes; however, the mechanisms regulating miR-183 in adipogenesis remain poorly understood. Here, we explored the expression pattern and role of miR-183 in adipogenesis using hircine preadipocytes. The results showed that miR-183 was up-regulated during preadipocyte differentiation, and overexpression of miR-183 enhanced lipid accumulation and dramatically increased the mRNA expression of the adipogenic genes PPARγ, C/EBPα, SREBP-1c, FAS, and ACC. Using bioinformatics tools, we predicted Smad4 to be a target of miR-183. This was subsequently validated with a luciferase reporter assay. Overexpression of miR-183 suppressed the mRNA and protein levels of Smad4 significantly, whereas inhibiting miR-183 had the opposite effect. However, inhibition of Smad4 greatly accelerated lipid deposition and increased the expression of adipogenic genes which consists with the results of miR-183 overexpression. In conclusion, these results indicate that miR-183 promotes hircine preadipocyte differentiation by targeting Smad4.

Introduction

Adipogenesis, a complex process in which fibroblast-like preadipocytes differentiate into lipid-laden and insulin-responsive mature adipocytes (Lefterova and Lazar, 2009), requires sequential activation of numerous transcription factors and noncoding RNAs. Among these, peroxisome proliferator-activated receptor γ (PPARγ) (Tontonoz et al., 1994) and CCAAT/enhancer binding protein α (C/EBPα) (Yeh et al., 1995; Rosen et al., 2002), known as adipogenic master genes, promote adipogenesis by activating genes such as fatty acid-binding protein 4, fatty acid synthase, and lipoprotein lipase. Reversely, the differentiation of adipocytes is inhibited by Smad4 via the transforming growth factor-β (TGF-β) signaling pathway (Ignotz and Massagué, 1985; Choy et al., 2000). In addition, microRNAs, a class of short noncoding RNAs that are generally regarded to inhibit gene expression by binding to the 3′-UTR of target mRNAs (Lagos-Quintana et al., 2001; Bagga et al., 2005), have been confirmed to regulate lipid metabolism in animals. Studies on human preadipocytes have demonstrated that miR-27 impairs human adipocyte differentiation by targeting PPARγ (Kim et al., 2010), while miR-103 enhances adipogenesis by inhibiting PDK1 (Wilfred et al., 2007). In the mouse, the role of several miRNAs has been studied during 3T3-L1 adipocyte differentiation. MiR-210 and the miR-17-92 cluster promote the differentiation of preadipocytes (Wang et al., 2008; Liang et al., 2013), while miR-302a represses mouse adipogenesis (Jeong et al., 2014). Furthermore, miR-143 (Esau et al., 2004) and miR-375 (Ling et al., 2011) promote adipogenesis, whereas miR-124 (Qadir et al., 2014) and miR-199a-5p (Alexander et al., 2013) have the opposite effect.

MiR-183 is a well-conserved microRNA across many species from invertebrates to humans. Many studies have unveiled the functions of miR-183 in neurosensory development (Pierce et al., 2008), tumor cell migration (Lowery et al., 2010), non-small cell lung cancer development (Zhang et al., 2015), and inner ear development (Sacheli et al., 2009). In pigs, the expression of miR-183 is higher in the backfat of Meishan pigs (Chinese indigenous fatty pig) than in Large White pigs (lean breed of pig) (Chen et al., 2012). During 3T3-L1 pre-adipocyte differentiation, miR-183 levels are increased, which subsequently promotes adipogenesis by targeting low-density lipoprotein receptor-related protein 6 (LRP6) (Kajimoto et al., 2006; Chen et al., 2014). Given the many-to-many relationships between miRNAs and their targets (Sarver et al., 2010), the various mechanisms employed by miR-183 to regulate preadipocyte differentiation warrants investigation.

As an economically important animal, domestic goats primarily serve as a major source of meat. Adipose tissue is directly associated with the yield and quality of meat. Previous studies have revealed that miR-183 plays a positive role in mouse adipogenesis (Kajimoto et al., 2006; Chen et al., 2014); however, its function in goat adipocyte development remains unclear. In this study, we studied the roles and potential mechanisms of miR-183 in goat adipogenesis. Our results revealed that overexpression of miR-183 accelerated the expression of adipogenic marker genes and lipid accumulation during hircine preadipocyte differentiation, while repressing miR-183 attenuated lipid accumulation and adipogenic gene expression. We also validated Smad4 as a target gene of miR-183. Decreasing Smad4 promoted adipogenesis, yielding the same effect as the overexpression of miR-183. Collectively, these results unveiled that miR-183 plays a positive role in hircine preadipocyte differentiation via the suppression of Smad4, which clarifies the roles of miRNA in goat adipogenesis.

Section snippets

Cell isolation

All of the experimental procedures for this experiment were conducted under a protocol approved by the Institutional Animal Care and Use Committee in the College of Animal Science and Technology, Sichuan Agricultural University, China. Hircine preadipocytes were separated from the cervical subcutaneous adipose tissue of 3-day-old Nanjiang Brown goats under sterile conditions. Subcutaneous adipose tissue was rinsed three times in PBS then minced and digested with 1 mg/mL of collagenase type I

The expression profile of miR-183 during goat preadipocyte differentiation

Immunoassay analyses revealed that the isolated preadipocytes were positive for adipocyte protein 2 (AP2) (Fig. 1a). After eight days of induction, the adipocytes were fully differentiated, depositing large lipid droplets (Fig. 1b). Simultaneously, the expression of adipogenic marker gene PPARγ accumulated and peaked on the sixth day of induction (Fig. 1c). Correspondingly, miR-183 expression rapidly increased by approximately three-fold after two days of induction and then decreased to the

Discussion

Adipocyte differentiation is a complex process regulated at different levels by various factors (Rosen and MacDougald, 2006; Christodoulides et al., 2009; Karbiener et al., 2009). Among these factors, miRNAs were previously shown to regulate adipocyte differentiation by binding to the 3′-UTR of adipogenesis-related-genes (Xie et al., 2009a; Xie et al., 2009b). During 3T3-L1 preadipocyte differentiation, miR-183 promotes adipogenesis by targeting LRP6 (Kajimoto et al., 2006; Chen et al., 2014).

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 31772578) and the Science and Technology Program of Sichuan Province, China (No. 2016NYZ0045). We thank Natasha Beeton-Kempen, PhD, from Liwen Bianji, Edanz Editing China, for editing the English text of a draft of this manuscript.

References (38)

  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method

    Methods

    (2001)
  • R. Sacheli et al.

    Expression patterns of miR-96, miR-182 and miR-183 in the development inner ear

    Gene Expr. Patterns

    (2009)
  • P. Tontonoz et al.

    Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor

    Cell

    (1994)
  • Bernard R. Wilfred et al.

    Energizing miRNA research: a review of the role of miRNAs in lipid metabolism, with a prediction that miR-103/107 regulates human metabolic pathways

    Mol. Genet. Metab.

    (2007)
  • H.F. Xu et al.

    Overexpression of SREBP1 (sterol regulatory element binding protein 1) promotes de novo fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells

    J. Dairy Sci.

    (2016)
  • M.S. Alexander et al.

    MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation

    Cell Death Differ.

    (2013)
  • C. Chen et al.

    Solexa sequencing identification of conserved and novel microRNAs in backfat of Large White and Chinese Meishan pigs

    PLoS One

    (2012)
  • L. Choy et al.

    Roles of autocrine TGF-β receptor and Smad signaling in adipocyte differentiation

    J. Cell Biol.

    (2000)
  • R.A. Ignotz et al.

    Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts

    Proc. Natl. Acad. Sci. U. S. A.

    (1985)
  • Cited by (14)

    • CircBTBD7 inhibits adipogenesis via the miR-183/SMAD4 axis

      2023, International Journal of Biological Macromolecules
    • The cross-talk between adipokines and miRNAs in health and obesity-mediated diseases

      2019, Clinica Chimica Acta
      Citation Excerpt :

      Previously, the role of miRNAs in adipogenesis during obesity has been reviewed by other groups [33,40–42,63–65]. Table 1 presents a summary of recent studies published in 2018 [66–94] and 2019 [95–104] in this field. Although the mechanisms underlying the functions of miRNAs in obesity are not completely clear, several studies have proposed that adipokines represent a major modulator of obesity-mediated changes in miRNA expression in human tissues, with effects on health and diseases, which are reviewed in the next sections Fig. 3.

    View all citing articles on Scopus
    1

    Contributed equally to this work.

    View full text