Tian Weihua, Liu Ye, Zhang Wenhui, Nie Ruixue, Ling Yao, Zhang Bo, Zhang Hao, Wu Changxin
National Engineering Laboratory for Animal Breeding, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China.
J Anim Sci Biotechnol. 2023 Jul 6;14(1):91. doi: 10.1186/s40104-023-00891-8.
Abdominal fat deposition depends on both the proliferation of preadipocytes and their maturation into adipocytes, which is a well-orchestrated multistep process involving many regulatory molecules. Circular RNAs (circRNAs) have emergingly been implicated in mammalian adipogenesis. However, circRNA-mediated regulation in chicken adipogenesis remains unclear. Our previous circRNA sequencing data identified a differentially expressed novel circRNA, 8:27,886,180|27,889,657, during the adipogenic differentiation of chicken abdominal preadipocytes. This study aimed to investigate the regulatory role of circDOCK7 in the proliferation and adipogenic differentiation of chicken abdominal preadipocytes, and explore its molecular mechanisms of competing endogenous RNA underlying chicken adipogenesis.
Our results showed that 8:27,886,180|27,889,657 is an exonic circRNA derived from the head-to-tail splicing of exons 19-22 of the dedicator of cytokinesis 7 (DOCK7) gene, abbreviated as circDOCK7. CircDOCK7 is mainly distributed in the cytoplasm of chicken abdominal preadipocytes and is stable because of its RNase R resistance and longer half-life. CircDOCK7 is significantly upregulated in the abdominal fat tissues of fat chickens compared to lean chickens, and its expression gradually increases during the proliferation and adipogenic differentiation of chicken abdominal preadipocytes. Functionally, the gain- and loss-of-function experiments showed that circDOCK7 promoted proliferation, G0/G1- to S-phase progression, and glucose uptake capacity of chicken abdominal preadipocytes, in parallel with adipogenic differentiation characterized by remarkably increased intracellular lipid droplet accumulation and triglyceride and acetyl coenzyme A content in differentiated chicken abdominal preadipocytes. Mechanistically, a pull-down assay and a dual-luciferase reporter assay confirmed that circDOCK7 interacted with gga-miR-301b-3p, which was identified as an inhibitor of chicken abdominal adipogenesis. Moreover, the ACSL1 gene was demonstrated to be a direct target of gga-miR-301b-3p. Chicken ACSL1 protein is localized in the endoplasmic reticulum and mitochondria of chicken abdominal preadipocytes and acts as an adipogenesis accelerator. Rescue experiments showed that circDOCK7 could counteract the inhibitory effects of gga-miR-301b-3p on ACSL1 mRNA abundance as well as the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.
CircDOCK7 serves as a miRNA sponge that directly sequesters gga-miR-301b-3p away from the ACSL1 gene, thus augmenting adipogenesis in chickens. These findings may elucidate a new regulatory mechanism underlying abdominal fat deposition in chickens.
腹部脂肪沉积取决于前脂肪细胞的增殖及其向脂肪细胞的成熟，这是一个精心编排的多步骤过程，涉及许多调节分子。环状RNA（circRNA）已逐渐被证明与哺乳动物脂肪生成有关。然而，circRNA介导的鸡脂肪生成调控仍不清楚。我们之前的circRNA测序数据在鸡腹部前脂肪细胞的成脂分化过程中鉴定出一种差异表达的新型circRNA，8:27,886,180|27,889,657。本研究旨在探讨circDOCK7在鸡腹部前脂肪细胞增殖和成脂分化中的调控作用，并探索其在鸡脂肪生成中作为竞争性内源RNA的分子机制。
我们的结果表明，8:27,886,180|27,889,657是一种外显子circRNA，由胞质分裂7（DOCK7）基因的第19 - 22外显子头对头拼接而成，简称为circDOCK7。CircDOCK7主要分布在鸡腹部前脂肪细胞的细胞质中，由于其对RNase R具有抗性且半衰期较长而较为稳定。与瘦鸡相比，circDOCK7在肥鸡的腹部脂肪组织中显著上调，并且在鸡腹部前脂肪细胞的增殖和成脂分化过程中其表达逐渐增加。在功能上，功能获得和功能缺失实验表明，circDOCK7促进了鸡腹部前脂肪细胞的增殖、从G0/G1期到S期的进展以及葡萄糖摄取能力，同时促进了成脂分化，其特征是分化的鸡腹部前脂肪细胞内脂质滴积累以及甘油三酯和乙酰辅酶A含量显著增加。机制上，下拉实验和双荧光素酶报告实验证实circDOCK7与gga - miR - 301b - 3p相互作用，gga - miR - 301b - 3p被鉴定为鸡腹部脂肪生成的抑制剂。此外，ACSL1基因被证明是gga - miR - 301b - 3p的直接靶标。鸡ACSL1蛋白定位于鸡腹部前脂肪细胞的内质网和线粒体中，并作为脂肪生成促进因子发挥作用。拯救实验表明，circDOCK7可以抵消gga - miR - 301b - 3p对ACSL1 mRNA丰度以及鸡腹部前脂肪细胞增殖和成脂分化的抑制作用。
CircDOCK7作为一种miRNA海绵，直接将gga - miR - 301b - 3p从ACSL1基因上隔离，从而增强鸡的脂肪生成。这些发现可能阐明了鸡腹部脂肪沉积的一种新的调控机制。
