Research paperMiR-183 promotes preadipocyte differentiation by suppressing Smad4 in goats
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)
- et al.
Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation
Cell
(2005) - et al.
Mature miR-183, negatively regulated by transcription factor GATA3, promotes 3T3-L1 adipogenesis through inhibition of the canonical Wnt/β-catenin signaling pathway by targeting LRP6
Cell. Signal.
(2014) - et al.
Transforming growth factor-β inhibits adipocyte differentiation by Smad3 interacting with CCAAT/enhancer-binding protein (C/EBP) and repressing C/EBP transactivation function
J. Biol. Chem.
(2003) - et al.
Adipogenesis and WNT signalling
Trends Endocrinol. Metab.
(2009) - et al.
MicroRNA-143 regulates adipocyte differentiation
J. Biol. Chem.
(2004) Transcriptional control of adipocyte formation
Cell Metab.
(2006)- et al.
MicroRNA-302a inhibits adipogenesis by suppressing peroxisome proliferator-activated receptor γ expression[J]
FEBS Lett.
(2014) - et al.
microRNA miR-27b impairs human adipocyte differentiation and targets PPARγ
Biochem. Biophys. Res. Commun.
(2009) - et al.
miR-27a is a negative regulator of adipocyte differentiation via suppressing PPARgamma expression
Biochem. Biophys. Res. Commun.
(2010) - et al.
New developments in adipogenesis
Trends Endocrinol. Metab.
(2009)
Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method
Methods
Expression patterns of miR-96, miR-182 and miR-183 in the development inner ear
Gene Expr. Patterns
Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor
Cell
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.
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.
MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation
Cell Death Differ.
Solexa sequencing identification of conserved and novel microRNAs in backfat of Large White and Chinese Meishan pigs
PLoS One
Roles of autocrine TGF-β receptor and Smad signaling in adipocyte differentiation
J. Cell Biol.
Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts
Proc. Natl. Acad. Sci. U. S. A.
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