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.

中文翻译：

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MiR-9-5p通过抑制瘦素基因表达来促进兔前脂肪细胞分化。

微小RNA（miRNA）是一类小的非编码RNA，它们参与细胞分化的调控。先前的研究表明，miR-9-5p在癌细胞的发育中起着关键作用，但对miR-9-5p调节脂肪形成的机制仍然知之甚少。本研究旨在通过观察miR-9-5p在前脂肪细胞中的调节作用并寻找相关的靶标来研究其在生产优质肉兔中的意义。在这项研究中，采用了双重荧光素酶报告基因分析来验证miR-9-5p和瘦素基因之间的靶向关系。我们还利用了定量逆转录PCR（qRT-PCR），蛋白质印迹，油红O染色测定，和甘油三酸酯含量的测定，以分析miR-9-5p和瘦素基因在脂肪细胞分化过程中的调控。分析表明，在脂肪前细胞分化过程中，miR-9-5p被上调并且与脂肪形成相关的生物标志物，即脂肪酸结合蛋白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）也被上调。同时，油红O染色法显示脂质滴的积累增加。我们还探讨了使用白色前脂肪细胞的miR-9-5p在脂肪形成中的表达模式和作用。结果表明，miR-9-5p在前脂肪细胞分化过程中被上调，miR-9-5p的过表达增强了脂质的积累。此外，我们发现miR-9-5p的过表达显着上调了标记基因PPARγ，C /EBPα和FABP4的表达，并增加了PPARγ的蛋白质水平和甘油三酸酯含量。结果表明，miR-9-5p可能参与了家兔前脂肪细胞分化的调控。通过使用生物信息学工具，我们预测瘦蛋白是miR-9-5p的靶基因，并且该结论已通过荧光素酶报告基因检测法得到验证。最后，我们证实了si-瘦素对瘦素的抑制作用促进了家兔前脂肪细胞的分化。本研究的结果表明，miR-9-5p通过靶向瘦素基因来调节家兔的白色脂肪细胞分化。结果表明，miR-9-5p可能参与了家兔前脂肪细胞分化的调控。通过使用生物信息学工具，我们预测瘦蛋白是miR-9-5p的靶基因，并且该结论已通过荧光素酶报告基因检测法得到验证。最后，我们证实了si-瘦素对瘦素的抑制作用促进了兔前脂肪细胞的分化。本研究的结果表明，miR-9-5p通过靶向瘦素基因来调节家兔的白色脂肪细胞分化。结果表明，miR-9-5p可能参与了家兔前脂肪细胞分化的调控。通过使用生物信息学工具，我们预测瘦蛋白是miR-9-5p的靶基因，并且该结论已通过荧光素酶报告基因检测法得到验证。最后，我们证实了si-瘦素对瘦素的抑制作用促进了兔前脂肪细胞的分化。本研究的结果表明，miR-9-5p通过靶向瘦素基因来调节家兔的白色脂肪细胞分化。我们证实了si-瘦素对瘦素的抑制作用促进了兔前脂肪细胞的分化。本研究的结果表明，miR-9-5p通过靶向瘦素基因来调节家兔的白色脂肪细胞分化。我们证实了si-瘦素对瘦素的抑制作用促进了兔前脂肪细胞的分化。本研究的结果表明，miR-9-5p通过靶向瘦素基因来调节家兔的白色脂肪细胞分化。