ABSTRACT

Porcine fat deposition is an important economic trait of pig breeds, and understanding the gene regulatory network in adipocytes is essential for modern pig breeding. In a previous study, we demonstrated that miR-29a negatively regulates the differentiation of porcine adipocytes. In this study, we investigated the roles of miR-29b/c in porcine adipocytes and the underlying mechanisms. Using EdU staining and the CCK-8 assay, we observed that transfection with the miR-29b/c agomir promoted the proliferation of porcine intramuscular (IM) and subcutaneous (SC) adipocytes by altering the expression of cell-cycle-related genes. According to the results of oil red O staining and western blot analysis, transfection with the miR-29b/c agomir suppressed the differentiation of porcine SC and IM adipocytes via the AKT/PKA/MAPK signalling pathway. Furthermore, we proved that miR-29b/c regulates porcine adipocytes by directly targeting the 3ʹ-untranslated region (3ʹUTR) of CTRP6 using a dual-luciferase reporter assay. Finally, co-transfection with miR-29b/c and CTRP6 partially restored the changes of phenotype and gene expression induced by miR-29b/c overexpression in 3T3-L1 adipocytes. Taken together, our data demonstrate that both miR-29b and miR-29 c negatively regulate porcine adipogenesis by targeting CTRP6, which furthers our understanding of the gene network that regulates fat deposition in pigs.

摘要

猪脂肪沉积是猪品种的重要经济性状，了解脂肪细胞中的基因调控网络对于现代猪育种至关重要。在之前的一项研究中，我们证明 miR-29a 负向调节猪脂肪细胞的分化。在这项研究中，我们研究了 miR-29b/c 在猪脂肪细胞中的作用及其潜在机制。使用 EdU 染色和 CCK-8 测定，我们观察到转染 miR-29b/c agomir 通过改变细胞周期相关基因的表达促进猪肌肉内 (IM) 和皮下 (SC) 脂肪细胞的增殖。根据油红 O 染色和蛋白质印迹分析的结果，转染 miR-29b/c agomir 通过 AKT/PKA/MAPK 信号通路抑制了猪 SC 和 IM 脂肪细胞的分化。此外，我们证明 miR-29b/c 通过使用双荧光素酶报告基因检测直接靶向 CTRP6 的 3ʹ-非翻译区 (3ʹUTR) 来调节猪脂肪细胞。最后，与 miR-29b/c 和 CTRP6 共转染部分恢复了 3T3-L1 脂肪细胞中由 miR-29b/c 过表达诱导的表型和基因表达的变化。总之，我们的数据表明 miR-29b 和 miR-29c 通过靶向 CTRP6 对猪脂肪生成负调控，这进一步加深了我们对调控猪脂肪沉积的基因网络的理解。与 miR-29b/c 和 CTRP6 共转染部分恢复了 3T3-L1 脂肪细胞中由 miR-29b/c 过表达诱导的表型和基因表达的变化。总之，我们的数据表明 miR-29b 和 miR-29c 通过靶向 CTRP6 对猪脂肪生成负调控，这进一步加深了我们对调控猪脂肪沉积的基因网络的理解。与 miR-29b/c 和 CTRP6 共转染部分恢复了 3T3-L1 脂肪细胞中由 miR-29b/c 过表达诱导的表型和基因表达的变化。总之，我们的数据表明 miR-29b 和 miR-29c 通过靶向 CTRP6 对猪脂肪生成负调控，这进一步加深了我们对调控猪脂肪沉积的基因网络的理解。