miR-542-3p reduces antioxidant capacity in goat caput epididymal epithelial cells by targeting glutathione peroxidase 5 (GPx5)

Abstract

The mammalian epididymis provides an optimal and antioxidative fluid microenvironment for post-testicular sperm maturation by secretion of antioxidant scavengers and removal of excessive ROS. MicroRNAs (miRNAs) are expressed in the epididymis and involved in the regulation of epididymis physiology and functions. However, whether miRNAs are involved in regulating the antioxidant capacity and oxidative damage in the epididymis is not well understood. This study was designed to investigate the role of miR-542-3p in the regulation of antioxidant capacity and oxidative damage in the epididymis of goats. Firstly, we determined the expression of miR-542-3p and glutathione peroxidase 5 (GPx5) in the epididymis of young and adult goats using RT-qPCR assay, and found that miR-542-3p and GPx5 exhibited inverse expression levels. Our results showed that the expression level of miR-542-3p in epididymis was upregulated (P < 0.05) in young goats compared to adult goats, whereas the expression level of GPx5 in epididymis was downregulated (P < 0.01) in young goats compared to adult goats. Next, we further investigated the roles and potential mechanisms of miR-542-3p in epididymis using goat caput epididymal epithelial cells (GCEECs) isolated from Tai-hang goats (9-month-old). Our results showed that the overexpression of miR-542-3p in GCEECs transfected with miR-542-3p mimics resulted in decreased (P < 0.05) antioxidant enzyme activities of superoxide dismutase (SOD) and catalase (CAT). Similarly, the overexpression of miR-542-3p in GCEECs resulted in decreased (P < 0.05) glutathione (GSH) content and total antioxidant capacity (TAOC). In addition, the overexpression of miR-542-3p in GCEECs resulted in increased (P < 0.05) malonaldehyde (MDA) content. The inverse results of SOD, CAT, GSH, TAOC and MDA were observed in the down-expression of miR-542-3p in GCEECs transfected with miR-542-3p inhibitors (P < 0.05). Furthermore, GPx5 was confirmed to be a validated target of miR-542-3p in GCEECs using a dual-luciferase reporter assay, and transfection of miR-542-3p mimics decreased (P < 0.05) the mRNA expression and protein level of GPx5. In conclusion, our results indicated that miR-542-3p reduced antioxidant capacity and increased oxidative damage in GCEECs by targeting GPx5. The present study further understands the regulation of antioxidant capacity and epididymal-specific GPx5 secretion in caput epididymidis.

Keywords: Antioxidant capacity; Caput epididymal epithelial cell; GPx5; Oxidative damage; miR-542–3p.