College of Animal Science, Shanxi Agricultural University, Taigu 030801, China.
Royal Veterinary College, University of London, London NW1 0TU, UK.
Domest Anim Endocrinol. 2022 Jul;80:106711. doi: 10.1016/j.domaniend.2022.106711. Epub 2022 Feb 5.
Fat content is an important index to evaluate the individual performance of livestock animals such as sheep for meat production purposes. Reducing the subcutaneous and visceral fat while increasing the intramuscular fat is a valuable goal to achieve for the meat production industry. Here, we investigated the effect of miR-128-1-5p on adipogenesis of subcutaneous fat by targeting 5'-UTR in KLF11, a rare mechanism where most miRNAs bind the 3'-UTR of mRNAs. A dual fluorescence reporter assay was conducted to validate the binding sites of miR-128-1-5p on 5'-UTR of KLF11 mRNA. Roles of miR-128-1-5p in KLF11 expression were measured through co-transfecting miRNA mimics with KLF11-expressing vectors (CDSs together with or without the 5'-UTR) into ovine stromal vascular fractions (SVF). Additionally, functional roles of miR-128-1-5p, and KLF11 in adipogenesis of ovine subcutaneous fat were investigated. Results showed that miR-128-1-5p targeted KLF11 5'-UTR, reduced the fluorescence activity of the dual fluorescent reporter vector, as well as KLF11 mRNA, and protein expression levels. During the differentiation of SVF, disturbing the expression of miR-128-1-5p and KLF11 changed the adipogenic differentiation of SVF as observed in the lipid formation, and adipogenic marker genes. This study indicates that miR-128-1-5p promotes the expression of lipogenic marker genes and the formation of lipid droplets by targeting KLF11 5'-UTR. Furthermore, overexpression, and inhibition of KLF11 indicate that KLF11 inhibited SVF differentiation. In summary, the 5'-UTR binding mechanism discovered in this study extends the understanding of miRNA functions. Key roles of miR-128-1-5p and KLF11 in the adipogenesis of sheep subcutaneous fat have potential values for improving the meat and/or fat ratio of domestic animals.
脂肪含量是评估绵羊等家畜肉类生产个体性能的一个重要指标。减少皮下和内脏脂肪，同时增加肌内脂肪是肉类生产行业的一个有价值的目标。在这里，我们研究了 miR-128-1-5p 通过靶向 KLF11 的 5'-UTR 对皮下脂肪脂肪生成的影响，这是一种大多数 miRNA 结合 mRNA 3'-UTR 的罕见机制。进行了双荧光报告基因实验来验证 miR-128-1-5p 与 KLF11 mRNA 5'-UTR 的结合位点。通过将 miRNA 模拟物与 KLF11 表达载体（CDS 与或不与 5'-UTR 一起）共转染到绵羊基质血管部分（SVF）中来测量 miR-128-1-5p 在 KLF11 表达中的作用。此外，还研究了 miR-128-1-5p 和 KLF11 在绵羊皮下脂肪脂肪生成中的功能作用。结果表明，miR-128-1-5p 靶向 KLF11 的 5'-UTR，降低了双荧光报告载体的荧光活性，以及 KLF11 mRNA 和蛋白表达水平。在 SVF 分化过程中，干扰 miR-128-1-5p 和 KLF11 的表达改变了 SVF 的成脂分化，如脂质形成和成脂标记基因观察到的那样。这项研究表明，miR-128-1-5p 通过靶向 KLF11 的 5'-UTR 促进脂肪生成标记基因的表达和脂质滴的形成。此外，KLF11 的过表达和抑制表明 KLF11 抑制了 SVF 的分化。总之，本研究发现的 5'-UTR 结合机制扩展了对 miRNA 功能的认识。miR-128-1-5p 和 KLF11 在绵羊皮下脂肪脂肪生成中的关键作用对于提高家畜的肉/脂肪比具有潜在价值。
