MiRNA-375 promotes 3T3-L1 adipocyte differentiation via modulation of ERK signaling

Adipocyte hypertrophy and hyperplasia are important processes in the development of obesity. To understand obesity and its associated diseases, it is important to elucidate the molecular mechanisms governing adipogenesis. MiR-375 has been demonstrated to inhibit differentiation of neurites and participate in the regulation of insulin secretion and blood homeostasis. However, it is unknown whether miR-375 plays a role in adipocyte differentiation. To investigate the role of miR-375 in adipocyte differentiation, we compared miR-375 expression level between 3T3-L1 pre-adipocytes and adipocytes using miRNA microarray and quantitative real-time RT-PCR (qRT-PCR) analysis. Furthermore, we evaluated the effects of overexpression or inhibition of miR-375 on 3T3-L1 adipocyte differentiation. In this study, we found that miR-375 expression was increased after induction of adipogenic differentiation. Overexpression of miR-375 enhanced 3T3-L1 adipocyte differentiation: as evidenced by its ability to increase mRNA levels of both CCAAT/enhancer binding proteinα (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ2) and induction of adipocyte fatty acid-binding protein (aP2) and triglyceride (TG) accumulation. Furthermore, we found overexpression of miR-375 suppressed phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2). In contrast, Anti-miR-375 increased ERK1/2 phosphorylation levels and inhibited mRNA expression of C/EBPα, PPARγ2 and aP2 in 3T3-L1 adipocyte, accompanied by decreased adipocyte differentiation. Taken together, these data suggest that miR-375 promotes 3T3-L1 adipocyte differentiation, possibly via modulating ERK - PPARγ2 - aP2 pathway. Keywords: MiRNA-375, 3T3-L1 Adipocytes, Differentiation, Obesity, ERK1/2 INTRODUCTION Epidemic obesity is now a major health challenge in both developed and developing countries.1 The excessive growth, differentiation and hypertrophy of adipocytes, the major cellular component in fat tissue, are fundamental processes in the development of obesity. 2 Thus adipocytes are emerging as major targets in the prevention and treatment of obesity, type 2 diabetes, and cardiovascular disease. 3 To this end, it is important to understand the molecular mechanisms governing adipogenesis. The mouse 3T3-L1 pre-adipocyte cell line is a widely-used adipocyte differentiation model. Upon treatment with a standard cocktail of hormones, the cells undergo growth arrest, clonal expansion and subsequent terminal differentiation into mature adipocytes. This process is characterized by the expression of adipocyte-specific proteins such as aP2 protein, which is under the control of two key transcription factors: C/EBPα and PPARγ. 4 PPARγ is one of the nuclear receptors and is phosphorylated at its Ser84 site by ERKs. The ERK-PPARγ pathway is thought to play a role in adipocyte differentiation. 5–6 MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate gene expression. 7 MiRNAs have been involved in numerous physiological and pathological processes, including development,8–9 energy homeostasis,10 sugar and lipid metabolism,11–13 and tumorigenesis.8,14 Recent findings indicate some miRNAs play a role in regulating adipocyte differentiation. For instance, miR-143 was up-regulated after induction of differentiation in human pre-adipocytes and mouse 3T3-L1 cells, and antisense oligonucleotides against miR-143 blocked adipocyte differentiation.15–16 The miR-103 and miR-17/92 cluster accelerated adipocyte differentiation, 17–18 while let-7, miR-27 and miR-27b impaired adipogenesis.19–21 However, it is unknown whether miR-375 plays a role in adipocyte differentiation. Six years ago, miR-375 was identified as a pancreatic-specific miRNA implicated in the regulation of insulin secretion.22 Recent studies further demonstrated that miR-375 is also important in maintaining blood homeostasis and inhibiting neurite differentiation. 23–25 Based on these observations, we sought to test the hypothesis that miR-375 is important in the regulation of adipocyte differentiation using an in vitro 3T3-L1 pre-adipocyte model.