MicroRNA-129-1-3p protects chicken granulosa cells from cadmium-induced apoptosis by down-regulating the MCU-mediated Ca2+ signaling pathway

Abstract

Cadmium (Cd) is known as a female reproductive toxicant. Our previous study has shown that Cd can influence the proliferation and cell cycle of granulosa cells and induce apoptosis. MicroRNAs (miRNAs) play an important role in the regulation of Cd-induced granulosa cell damage in chickens. However, the mechanism remains unclear. In this study, we investigated the mechanisms by which microRNA-129–1-3p (miR-129–1-3p) regulates Cd-induced cytotoxicity in chicken granulosa cells. As anticipated, exposure to Cd resulted in the induction of oxidative stress in granulosa cells, accompanied by the downregulation of antioxidant molecules and/or enzymes of Nrf2, Mn-SOD, Cu-Zn SOD and CAT, and the upregulation of Keap1, GST, GSH-Px, GCLM, MDA, hydrogen peroxide and mitochondrial reactive oxygen species (mtROS). Further studies found that Cd exposure causes mitochondrial calcium ions (Ca2+) overload, provoking mitochondrial damage and apoptosis by upregulating IP3R, GRP75, VDAC1, MCU, CALM1, MFF, caspase 3, and caspase 9 gene and/or protein expressions and mitochondrial Ca2+ levels, while downregulating NCX1, NCLX and MFN2 gene and/or protein expressions and mitochondrial membrane potential (MMP). The Ca2+ chelator BAPTA-AM or the MCU inhibitor MCU-i4 significantly rescued Cd-induced mitochondrial dysfunction, thereby attenuating apoptosis. Additionally, a luciferase reported assay and western blot analysis confirmed that miR-129–1-3p directly target MCU. MiR-129–1-3p overexpression almost completely inhibited protein expression of MCU, increased the gene and protein expressions of NCLX and MFN2 downregulated by Cd, and attenuated mitochondrial Ca2+ overload, MMP depression and mitochondria damage induced by Cd. Moreover, the overexpression of miR-129–1-3p led to a reduction in mtROS and cell apoptosis levels, and a suppression of the gene and protein expressions of caspase 3 and caspase 9. As above, these results provided the evidence that IP3R-MCU signaling pathway activated by Cd plays a significant role in inducing mitochondrial Ca2+ overload, mitochondrial damage, and apoptosis. MiR-129–1-3p exerts a protective effect against Cd-induced granulosa cell apoptosis through the direct inhibition of MCU expression in the ovary of laying hens.