Abstract
BackgroundMicroRNAs (miRNAs) are a class of small non-coding RNAs, which participate in the regulation of cell differentiation. Previous studies have demonstrated that miR-9-5p plays a key role in cancer cell development, but the mechanisms by which miR-9-5p regulates adipogenesis remain poorly understood. The present study intended to investigate its significance in producing rabbits with high-quality meat by observing the regulatory effect of miR-9-5p in preadipocytes and finding the related targets.MethodsIn this study, a dual-luciferase reporter assay was employed to validate the targeting relationship between miR-9-5p and leptin gene. We also utilized quantitative reverse transcription PCR (qRT-PCR), western blot, oil red-O staining assay, and determination of triglyceride content to analyze the regulation of miR-9-5p and leptin gene during adipocyte differentiation.ResultsThe analysis demonstrated that during preadipocyte differentiation, miR-9-5p was up-regulated and the fat formation related biomarkers, i.e., fatty acid-binding protein 4 (FABP4), CCAAT-enhancer binding protein α (C/EBPα), and peroxisome proliferator activated receptor γ (PPARγ) were also up-regulated. Meanwhile, the oil red-O staining assay revealed that the accumulation of lipid droplets increased. We also explored the expression pattern and role of miR-9-5p in adipogenesis using white pre-adipocytes. The results showed that miR-9-5p was up-regulated during preadipocyte differentiation, and overexpression of miR-9-5p enhanced lipid accumulation. Furthermore, we found that the overexpression of miR-9-5p significantly up- regulated the expression of marker genes, PPARγ, C/EBPα and FABP4, and increased the protein levels of PPARγ and triglyceride content. The results suggest that miR-9-5p might be involved in the regulation of rabbit preadipocyte differentiation. We predicted that leptin is the target gene of miR-9-5p, by using bioinformatics tools and the conclusion was validated by a luciferase reporter assay. Finally, we verified that the knock-down of leptin by si-leptin promoted preadipocyte differentiation in rabbits.ConclusionThe results of the present study indicate that miR-9-5p regulates white preadipocyte differentiation in rabbits by targeting the leptin gene.
Highlights
Adipose tissue, which may be brown, beige, or white, plays an important role in energy metabolism by generating heat and storing lipids and serve as an endocrine organ
Results of oil red-O staining showed that lipid droplets rapidly increased during preadipocyte cell differentiation (Fig. 1b)
Upregulation of miR-9-5p promotes rabbit preadipocyte differentiation In order to investigate the function of miR-9-5p in rabbit preadipocyte cell differentiation, we first detected the expression level of miR-9-5p in the cells transfected with miR-9-5p mimics and Negative Control (NC) during differentiation
Summary
Adipose tissue, which may be brown, beige, or white, plays an important role in energy metabolism by generating heat and storing lipids and serve as an endocrine organ. Adipogenesis, is a complex process in which fibroblast-like preadipocytes differentiate into lipid-laden and insulin-responsive adipocytes [18] This process requires sequential activation of numerous transcription factors and noncoding RNAs. This process requires sequential activation of numerous transcription factors and noncoding RNAs Several transcription factors such as peroxisome proliferator-activated receptor-γ (PPARγ) and members of the CCAAT/enhancer-binding family of proteins (C/ EBPα) play a significant role in the regulation of adipogenesis [12, 28]. Leptin plays a pivotal role in regulating food intake, energy expenditure, and fat deposition in mammals [20] It can promote lipolysis and thermogenesis by influencing the sympathetic nervous system [6]. Recent literature shows that leptin enhances the differentiation of osteoblasts and inhibits the differentiation of adipocytes in human bone marrow stromal cells [36] These findings indicate that leptin may act locally in adipose tissue to influence the formation of mature adipocytes. The present study intended to investigate its significance in producing rabbits with high-quality meat by observing the regulatory effect of miR9-5p in preadipocytes and finding the related targets
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