Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, 211#Huimin Road, Wenjiang, Chengdu, 611130, Sichuan, China.
Lipids Health Dis. 2020 Jun 5;19(1):126. doi: 10.1186/s12944-020-01294-8.
MicroRNAs (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.
In 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.
The 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.
The results of the present study indicate that miR-9-5p regulates white preadipocyte differentiation in rabbits by targeting the leptin gene.
MicroRNAs（miRNAs）是一类小型非编码 RNA，参与细胞分化的调节。先前的研究表明，miR-9-5p 在癌细胞发育中起关键作用，但 miR-9-5p 调节脂肪生成的机制仍知之甚少。本研究旨在通过观察 miR-9-5p 在预脂肪细胞中的调节作用并找到相关靶标，来探讨其在生产优质兔肉兔子中的意义。
在这项研究中，采用双荧光素酶报告基因实验验证 miR-9-5p 与瘦素基因之间的靶向关系。我们还利用定量逆转录 PCR（qRT-PCR）、western blot、油红 O 染色测定法和甘油三酯含量测定来分析 miR-9-5p 和瘦素基因在脂肪细胞分化过程中的调节作用。
分析表明，在预脂肪细胞分化过程中，miR-9-5p 上调，脂肪形成相关生物标志物，如脂肪酸结合蛋白 4（FABP4）、CCAAT 增强子结合蛋白α（C/EBPα）和过氧化物酶体增殖物激活受体γ（PPARγ）也上调。同时，油红 O 染色测定法显示脂质滴的积累增加。我们还使用白色前脂肪细胞探索了 miR-9-5p 在脂肪生成中的表达模式和作用。结果表明，miR-9-5p 在预脂肪细胞分化过程中上调，miR-9-5p 的过表达增强了脂质积累。此外，我们发现 miR-9-5p 的过表达显著上调了标记基因 PPARγ、C/EBPα 和 FABP4 的表达，并增加了 PPARγ 和甘油三酯含量的蛋白水平。结果表明，miR-9-5p 可能参与调节兔前脂肪细胞分化。我们使用生物信息学工具预测瘦素是 miR-9-5p 的靶基因，通过荧光素酶报告基因实验验证了这一结论。最后，我们验证了 si-leptin 敲低瘦素可促进兔前脂肪细胞分化。
本研究结果表明，miR-9-5p 通过靶向瘦素基因调节兔白色前脂肪细胞分化。
