Oxidative stress is the redox state mainly resulting from imbalanced generation and elimination of reactive oxygen species (ROS). ROS and/or their metabolites are components of cell signaling pathways and play important roles in cellular physiology and pathophysiology [1]. However, excessive ROS level may cause damage to DNA, proteins and lipids, which eventually results in various diseases, including cancers, inflammation, neurodegenerative diseases and cardiovascular diseases [2]. As the subcellular compartments for cell energy synthesis and metabolism, mitochondria are also the main locations for ROS production and elimination. Disruption or functional impairment of mitochondria can accelerate ROS production [3]. Therefore, maintaining mitochondrial biosynthesis and function is important for controlling the intracellular ROS level. Recent years have witnessed the achievement of ROS scavenging using various natural antioxidants, including omega-3 polyunsaturated fatty acids (ω-3 PUFAs) [4]. Eicosapentaenoic acid (EPA) is a kind of ω-3 PUFA and an important component of biofilms. It has been shown to regulate the intracellular ROS level by activating mitochondrial uncoupling [5]. Our previous study also showed that EPA improved the antioxidant defense of HepG2 cells by enhancing mitochondrial function and biogenesis [6]. However, the exact molecular mechanism underlying EPA's antioxidant reaction remains unclear.

As a major group of noncoding small RNAs, microRNAs (miRNAs) have been shown to be involved in oxidative stress control. For instance, Špaková et al. demonstrated that miR-210 reduction promoted mitochondrial respiratory activity and reduced ROS level in malignant melanoma cells [7]. Das et al. reported that miR-181c activated ROS production in cardiomyocyte mitochondria, which eventually caused myocardial damage [8]. EPA has been found to exert its biological activities via miRNA regulation [9]. However, the miRNA-mediated antioxidant mechanism of EPA remains largely unknown. Our preliminary study showed that 48 h of EPA treatment led to increased miR-494-5p expression in HepG2 cells (data not shown). Although the biological activity of miR-494-5p has rarely been studied, a series of studies have indicated that miR-494-3p functioned as a tumor suppressor and was associated with cellular oxidative stress control. For instance, Xiong et al. [10] showed that miR-494-3p promoted oxidative stress-induced neuronal death by downregulating parkinsonism associated deglycase (DJ-1). Wang et al. [11] demonstrated that miR-494-3p inhibition prevented apoptosis of human nucleus pulposus cells by targeting the JunD pathway. Sun et al. [12] also demonstrated that miR-494-3p prevented hypoxia-induced apoptosis in LO2 cells by activating the PI3K/Akt pathway. However, whether and how miR-494-5p is involved in apoptosis regulation and associated with the oxidative stress response remain to be elucidated.

Mitochondrial biosynthesis, oxidative phosphorylation and dynamic changes are closely associated with the maintenance of mitochondrial homeostasis, which plays a key role in cellular redox balance [13]. On the other hand, dysregulated ROS can cause mitochondrial gene mutations that may disrupt the electron transport chain, eventually leading to mitochondrial dysfunction [14]. A series of studies have indicated that mitochondrial dysfunction may cause oxidative stress-associated diseases, including neurodegenerative diseases, cancers and inflammation [15]. miR-494-3p was found to regulate mitochondrial biogenesis in skeletal muscle cells and adipocytes by affecting the transcriptional signaling pathway [16,17]. Therefore, addressing the mechanism underlying mitochondrial homeostatic regulation by miR-494-5p may also shed light on the function of this process in oxidative stress control.

To address whether and how miR-494-5p mediated the antioxidant reaction of EPA, the target gene of miR-494-5p was identified. The results showed that the mitochondrial elongation factor 1 (MIEF1) gene, an important regulator of mitochondrial dynamics, was a potential target gene of miR-494-5p. MIEF1 plays a key role in mitochondrial fusion and fission [18], which thereby may affect the cellular oxidative stress defense. The current study further investigated the effects of miR-494-5p and MIEF1 on oxidative stress control, and the results may provide insights into the molecular antioxidant mechanism of EPA and the epigenetic and/or inheritable regulatory mechanism of cellular stress defense.