MicroRNA-129-1-3p attenuates autophagy-dependent cell death by targeting MCU in granulosa cells of laying hens under H2O2-induced oxidative stress

Abstract

The present study aimed to investigate the mechanism of microRNA-129-1-3p (miR-129-1-3p) in regulating hydrogen peroxide (H₂O₂)-induced autophagic death of chicken granulosa cell by targeting mitochondrial calcium uniporter (MCU). The results indicated that the exposure of hens' ovaries to H₂O₂ 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 H₂O₂ 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 H₂O₂-induced changes. Furthermore, miR-129-1-3p overexpression in granulosa cells prevented the alterations induced by H₂O₂ 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.

Keywords: MCU; granulosa cell autophagy; laying hens; microRNA-129-1-3p; oxidative stress.