miR-489-3p Regulates the Oxidative Stress Response in the Liver and Gill Tissues of Hybrid Yellow Catfish (Pelteobagrus fulvidraco♀ × P. vachelli♂) Under Cu2+ Exposure by Targeting Cu/Zn-SOD

Abstract

Copper/zinc superoxide dismutase (Cu/Zn-SOD) plays critical roles in protecting cells and tissues against oxidative damage. Excessive copper ions (Cu²⁺) in water can damage the cells of aquatic organisms, leading to impaired growth and development and reduced antioxidant defenses. Many regulatory factors control the response to excess Cu²⁺. Among them, microRNAs (miRNAs) are important small RNAs that regulate the expression of their target genes and participate in the oxidative stress response. In the present study, we used bioinformatics and dual luciferase reporter gene analyses to demonstrate that the miR-489-3p of hybrid yellow catfish (Pelteobagrus fulvidraco♀ × P. vachelli♂) binds to the 3'-untranslated region (UTR) of its target gene, which encodes a Cu/Zn-SOD. The regulatory relationship between this miRNA and its target gene Cu/Zn-SOD was analyzed using qRT-PCR and luciferase activity assays. We also investigated the effect of the loss of miR-489-3p expression on the oxidative stress response of hybrid yellow catfish exposed to Cu²⁺. The Cu/Zn-SOD 3'UTR region was found to be fully complementary to positions 2-9 of the 5'-end seed region of miR-489-3p. The miR-489-3p expression levels were negatively related to Cu/Zn-SOD expression. Silencing of miR-489-3p up-regulated Cu/Zn-SOD expression in the liver and gill tissues, increased activities of SOD and catalase, and reduced the malondialdehyde content. This study is the first to demonstrate that miR-489-3p targets Cu/Zn-SOD to mediate the oxidative response to metal stress. These findings provide a theoretical basis for further studies on the response to oxidative stress caused by metals in cultured fish, and provide an experimental basis for the management of the culture environment.

Keywords: Cu/Zn-SOD; Cu2+; antioxidant system; hybrid yellow catfish (Pelteobagrus fulvidraco♀ × P. vachelli♂); miR-489-3p.

FIGURE 1

Expression analysis of Cu/Zn-SOD gene in different tissues of healthy hybrid yellow catfish (Pelteobagrus fulvidraco♀ × P. vachelli♂) (control) or yellow catfish subjected to 140 μg⋅L^-1 Cu²⁺ stress for 24 h. (A) Expression levels of Cu/Zn-SOD in different tissues of control group or 140 μg⋅L^-1 Cu²⁺-stressed group for 24 h as determined by qRT-PCR. Different lowercase letters indicate significant differences among different tissues in control group (P < 0.05, Duncan’s multiple comparison). Different uppercase letters indicate significant differences among different tissues in 140 μg⋅L^-1 Cu²⁺-stressed group at 24 h (P < 0.05, Duncan’s multiple comparison). ^∗ indicates significant differences between control group and 140 μg⋅L^-1 Cu²⁺-stressed group (P < 0.05, independent sample t test). (B) Semi-quantitative results of Cu/Zn-SOD transcript levels in different tissues of control group or 140 μg⋅L^-1 Cu²⁺-stressed group at 24 h as determined by agarose gel electrophoresis analysis.

FIGURE 2

Verification of binding sites of miRNAs to potential target genes using dual luciferase reporter system. (A) miRNA can be paired with the 3′-UTR of potential target gene mRNA. (B) HEK-293T cells in 96-well plates were co-transformed with constructed pGL-Cu/Zn-SOD 3′UTR (wt) or pGL-Cu/Zn-SOD mutant (six-base mutant) and miRNA mimic or miRNA negative control (NC) (Ctrl) using Lipofectamine 2000 transfection reagent. Different lowercase letters indicate significant differences among experimental groups (P < 0.05, Duncan’s multiple comparison).

FIGURE 3

Analysis of regulatory relationships between miR-489-3p and Cu/Zn-SOD in vitro. (A) Luciferase activities were analyzed by co-transfecting Cu/Zn-SOD-3′UTR and 50 nM or 100 nM miR-489-3p mimic or same dose of miR-489-3p NC into hepatocytes. (A-1) pEGFP-C1-3Flag-Cu/Zn-SOD-3′UTR and 100 nM miR-489-3p NC were constructed and co-transfected into hepatocytes. Fluorescence was analyzed at 48 h after transfection; (A-2) pEGFP-C1-3Flag-Cu/Zn-SOD-3′UTR and 50 nM miR-489-3p mimic were constructed and co-transfected into hepatocytes. Fluorescence was analyzed at 48 h after transfection; (A-3) pEGFP-C1-3Flag-Cu/Zn-SOD-3′UTR and 100 nM miR-489-3p mimic were constructed and co-transfected into hepato cytes. Fluorescence was analyzed at 48 h after transfection; (B) Transcript levels of Cu/Zn-SOD in hepatocyte level at 48 h as determined by qRT-PCR. Different lowercase letters indicate significant differences among experimental groups (P < 0.05, Duncan’s multiple comparison).

FIGURE 4

Analysis of regulatory relationships between miR-489-3p (A) and Cu/Zn-SOD (B) in vivo. Juvenile hybrid yellow catfish (P. fulvidraco♀ × P. vachelli♂) weighing about 15.5 ± 0.8 g were injected in tail vein with PBS (control), miR-489-3p (NC), miR-489-3p antagomir (dose, 50 mg⋅kg^-1 body weight). ^∗ indicates significant differences between values obtained before and after injection (P < 0.05, paired-samples t test). Different lowercase letters indicate significant differences among different treatments at each sampling point (P < 0.05, Duncan’s multiple comparison).

FIGURE 5

Expression levels of miR-489-3p and Cu/Zn-SOD gene in liver (A,B) and gill (C,D) of hybrid yellow catfish (P. fulvidraco♀ × P. vachelli♂). Juvenile yellow catfish weighing about 15.5 ± 0.8 g were injected in tail vein with PBS (control), miR-489-3p (NC), or miR-489-3p antagomir (dose, 50 mg⋅kg^-1 body weight) and monitored for 108 h. ^∗ indicates significant differences between values obtained before and after injection (P < 0.05, paired-samples t test). Different lowercase letters indicate significant differences among different treatments at each sampling point (P < 0.05, Duncan’s multiple comparison).

FIGURE 6

Effect of inhibition of miR-489-3p on activities/levels of hepatic Cu/Zn-SOD (A), CAT (B), GSH-Px (C), and MDA (D) in hybrid yellow catfish (P. fulvidraco♀ × P. vachelli♂) exposed to Cu²⁺. At 12 h after injection with PBS (control) or miR-489-3p antagomir, yellow catfish were subjected to acute Cu²⁺ exposure for 96 h. ^∗ indicates significant differences between values obtained before and after injection (P < 0.05, paired-samples t test). Different lowercase letters indicate significant differences among different treatments at each sampling point (P < 0.05, Duncan’s multiple comparison).

FIGURE 7

Effect of inhibition miR-489-3p on activities/levels of gill Cu/Zn-SOD (A), CAT (B), GSH-Px (C), and MDA (D) in hybrid yellow catfish (P. fulvidraco♀ × P. vachelli♂) exposed to Cu²⁺. At 12 h after injection of PBS (control) or miR-489-3p antagomir, yellow catfish were subjected to acute Cu²⁺ exposure for 96 h. ^∗ indicates significant differences between values obtained before and after injection (P < 0.05, paired-samples t test). Different lowercase letters indicate significant differences among different treatments at each sampling point (P < 0.05, Duncan’s multiple comparison).