School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China; Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212100, China.
Qingdao Animal Husbandry Workstation (Qingdao Institute of Animal Science and Veterinary Medicine), Qingdao, Shandong 266100, China.
Poult Sci. 2023 Oct;102(10):103006. doi: 10.1016/j.psj.2023.103006. Epub 2023 Aug 3.
The present study aimed to investigate the mechanism of microRNA-129-1-3p (miR-129-1-3p) in regulating hydrogen peroxide (HO)-induced autophagic death of chicken granulosa cell by targeting mitochondrial calcium uniporter (MCU). The results indicated that the exposure of hens' ovaries to HO resulted in a significant elevation in reactive oxygen species (ROS) levels, as well as the apoptosis of granulosa cells and follicular atresia. This was accompanied by an upregulation of glucose-regulated protein 75 (GRP75), voltage-dependent anion-selective channel 1 (VDAC1), MCU, mitochondria fission factor (MFF), microtubule-associated protein 1 light chain 3 (LC3) I, and LC3II expression, and a downregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and mitofusin-2 (MFN2) expression. In hens' granulosa cells, a luciferase reporter assay confirmed that miR-129-1-3p directly regulates MCU. The induction of oxidative stress through HO resulted in the activation of the permeability transition pore, an overload of calcium, depolarization of the mitochondrial membrane potential, dysfunction of mitochondria-associated endoplasmic reticulum membranes (MAMs), and ultimately, autophagic cell death. The overexpression of miR-129-1-3p effectively mitigated these HO-induced changes. Furthermore, miR-129-1-3p overexpression in granulosa cells prevented the alterations induced by HO in the expression of key proteins that play crucial roles in maintaining the integrity of MAMs and regulating autophagy, such as GRP75, VDAC1, MFN2, PTEN-induced kinase 1 (Pink1), and parkin RBR E3 ubiquitin-protein ligase (Parkin). Together, these in vitro- and in vivo-based experiments suggest that miR-129-1-3p protects granulosa cells from oxidative stress-induced autophagic cell death by downregulating the MCU-mediated mitochondrial autophagy. miR-129-1-3p/MCU calcium signaling pathway may act as a new target to alleviate follicular atresia caused by oxidative stress in laying hens.
本研究旨在探讨 microRNA-129-1-3p（miR-129-1-3p）通过靶向线粒体钙单向转运体（MCU）调节过氧化氢（HO）诱导的鸡颗粒细胞自噬性死亡的机制。研究结果表明，母鸡卵巢暴露于 HO 会导致活性氧（ROS）水平显著升高，以及颗粒细胞凋亡和卵泡闭锁。同时，葡萄糖调节蛋白 75（GRP75）、电压依赖性阴离子选择通道 1（VDAC1）、MCU、线粒体分裂因子（MFF）、微管相关蛋白 1 轻链 3（LC3）I 和 LC3II 的表达上调，而过氧化物酶体增殖物激活受体γ共激活因子 1α（PGC-1α）和融合蛋白 2（MFN2）的表达下调。在母鸡的颗粒细胞中，荧光素酶报告基因检测证实 miR-129-1-3p 可直接调控 MCU。HO 诱导的氧化应激导致通透性转换孔（PTP）的激活，钙离子超载，线粒体膜电位去极化，线粒体内质网膜（MAMs）功能障碍，最终导致细胞自噬性死亡。miR-129-1-3p 的过表达有效缓解了这些 HO 诱导的变化。此外，在颗粒细胞中过表达 miR-129-1-3p 可防止 HO 诱导的关键蛋白表达改变，这些蛋白在维持 MAMs 完整性和调节自噬中起着关键作用，如 GRP75、VDAC1、MFN2、PTEN 诱导激酶 1（Pink1）和 parkin RBR E3 泛素蛋白连接酶（Parkin）。综上所述，这些基于体外和体内的实验表明，miR-129-1-3p 通过下调 MCU 介导线粒体自噬来保护颗粒细胞免受氧化应激诱导的自噬性细胞死亡。miR-129-1-3p/MCU 钙信号通路可能成为缓解产蛋鸡卵泡闭锁的新靶点。
