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Elucidation of the mechanism of miR-122-5p in mediating FOXO3 injury and apoptosis of mouse cochlear hair cells induced by hydrogen peroxide

Unveiling the mechanism of miR-122-5p in the mediation of forkhead box O3 (FOXO3) in regards to cochlear hair cell damage provides an effective solution for the treatment of ear hearing disorders. An oxidative stress model using a mouse cochlear hair cell line (HEI-OC1) was established via hydrogen peroxide (H2O2). Then HEI-OC1 cells were transfected with miR-122-5p mimic, miR-122-5p inhibitor, and lentiviral vector FOXO3-WT/MUT. Cell viability and apoptosis rate were determined by MTT assay and flow cytometry. Reactive oxygen species (ROS) were observed by confocal laser scanning microscopy. Bcl-2, Bax, capase-3 and c-caspase-9 levels were quantified by western blot analysis and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Enzyme-linked immunosorbent assay (ELISA) was used to detect superoxide dismutase (SOD) and malondialdehyde (MDA) levels, and flow cytometry was performed to measure the mitochondrial membrane potential levels. In the HEI-OC1 oxidative stress model after transfection, the miR-122-5p level was decreased, whereas the FOXO3 level was increased, Moreover, the increased FOXO3 level diminished the cell viability, but promoted cell apoptosis. Apart from this, the Bcl-2 level was downregulated, while levels of Bax, c-caspase-3, c-caspase-9, ROS and MDA were upregulated. Meanwhile, the mitochondrial membrane potential level was also elevated. Overexpression of miR-122-5p was able to partially offset the effects of FOXO3 in the H2O2-treated HEI-OC1 cells. Collectively, miR-122-5p restrained the decrease in HEI-OC1 cell viability and apoptosis induced by treatment with H2O2.


Introduction
Cochlear hair cells, as the mechanoreceptors of the inner ear, are essential to auditory and vestibular function, the loss of which ultimately leads to permanent sensory deficits in mammals (1). Hearing loss, a very frequent sensory disorder in humans, is mainly attributable to cochlear hair cell damage caused by hazardous factors containing ototoxic pharmaceutical agents, excessive noise, aging and genetic disorders. Oxidative stress and high levels of reactive oxygen species (ROS) have an involvement with drug-and noise-induced, and age-related hearing injury, while cisplatin, aminoglycosides and continuous noise can result in high level of ROS production in cochlear hair cells, thereby inducing cell apoptosis (2). Cochlear hair cells do not spontaneously regenerate following loss or damage, due to the limitation of regenerative capacity of vestibular organs (3). Therefore, it will be of great significance to fathom out the pathways and molecular regulators involved in the pathogenesis of ROS-related hair cell cytotoxicity for the advancement of therapies toward functional restoration.
MicroRNAs (miRNAs/miRs), an important class of small non-coding RNAs, bind to target mRNAs and subsequently inhibit protein expression through mRNA degradation or translational inhibition (4). miRNAs play pivotal roles in various important biological processes as well as in the development and progression of various human diseases, where one miRNA can exert impacts on multiple target genes, and multiple miRNAs in turn can also synergistically act on one target gene (5,6). Several research studies have manifested that miR-122-5p is associated with many diseases, especially tumors or cancers, including colorectal cancer, melanoma, gastric cancer, lung cancer and cervical cancer (7)(8)(9)(10)(11)(12). In addition, plasma miRNA-122-5p has been identified as a potential biomarker for liver injury among chronic hepatitis B (CHB) patients with persistently normal alanine aminotransferase (PNALT) levels (13), and transient ischemic attack in rats (14). Zhou et al (15) demonstrated that miRNA-122-5p promotes the proliferation and DNA synthesis and represses the early apoptosis of human spermatogonial stem cells via targeting CBL and competing with lncRNA CASC7. Peng et al (16) reported that lncRNA XIST relieves hypoxia-induced injury in H9c2 cardiomyocytes via targeting the miR-122-5p/FOXP2 axis. Furthermore, many miRNAs also play vital roles in the development of cochlea inner ear hair cells and may be pivotal regulators in the process of hearing loss (17)(18)(19)(20)(21). Wang et al (2) demonstrated that tert-butyl hydroperoxide (t-BHP) promotes the production of ROS, and miR-122-5p expression was significantly downregulated in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. miR-122-5p was found to inhibit cell apoptosis and facilitate tumor progression by directly targeting forkhead box O3 (FOXO3) in α-fetoprotein (AFP)-producing gastric cancer (AFPGC) (7). Moreover, FOXO3 expression was found to be increased in the inner ear hair cells during cisplatin treatment in vitro (22). Therefore, we hypothesized that miR-122-5p can directly target FOXO3 to regulate the viability and apoptosis of cochlear hair cells under oxidative stress condition. The oxidative damage model was established in HEI-OC1 cells to elucidate the role and mechanism of miR-122-5p, hoping to provide more treatment options for hearing disorders. Our present study demonstrated that miR-122-5p overexpression attenuated the H 2 O 2 -induced damage in mouse cochlear hair cells by directly regulating FOXO3. Cell transfection. The miR-122-5p mimic (M; 5'-UGG AGU GAC AAU GGU GUU UG-3'), mimic control (MC; 5'-UUC UCC GAA CGU GUC ACG UTT-3') and FOXO3 lentivirus were obtained by transfection of 293T cells (ab266546, Abcam) with pPACKH1 Lentivector Packaging Kit (US SBI Co.). The FOXO3 overexpression vector was constructed by cloning the cDNA of mouse FOXO3 into the pPACKH1 lentivector. The HEI-OC1 cells were placed into a 24-well plate at the density of 1x10 5 cells/well. The density of the cells during lentiviral transfection was ~2x10 5 cells/well. The next day, the original medium was replaced by 2 ml of fresh medium containing 6 µg/ml polybrene, followed by the addition of an appropriate amount of the viral suspension. Subsequently, the membrane was incubated at 37˚C. After 4 h, another 2 ml of fresh medium was supplemented to dilute the polybrene. Following continuous culture for 24 h, the virus-containing medium was substituted with fresh medium, which was then used to continuously culture the cells. Four days later, the infected cells were collected by trypsinization and replated on a new 100-mm dish. While cell confluence reached about 30%, the culture medium was replaced by fresh medium containing 1 µg/µl puromycin for colony selection of stable transfected cells. The medium was changed every 3 day, and colonies were chosen and expanded for subsequent experiments after section. A blank vector lentivirus was used as a negative control (NC).

Materials and methods
Oxidative stress exposure and grouping. HEI-OC1 cells were exposed to 50 µM hydrogen peroxide (H 2 O 2 ) for 1 h post transfection to mimic oxidative stress condition, as previously reported (10). To unveil the effect of miR-122-5p in HEI-OC1 cells under oxidative stress, HEI-OC1 cells were assigned into four groups: Blank, Model, Model+MC and Model+M groups. Similarly, in order to explore the roles of miR-122-5p and FOXO3 in HEI-OC1 cells under oxidative stress, HEI-OC1 cells were divided into seven groups: Blank, Model, Model+MC, Model+M, Model+NC, Model+FOXO3 and Model+M+FOXO3 groups. The specific treatment of cells in the various groups was shown as follows. In the Blank group, HEI-OC1 cells were only incubated with medium; in the Model group, HEI-OC1 cells were exposed to 50 µM H 2 O 2 for 1 h; in the Model+MC group, HEI-OC1 cells were exposed to 50 µM H 2 O 2 for 1 h and then transfected with the mimic control; in the Model+M group, HEI-OC1 cells were exposed to 50 µM H 2 O 2 for 1 h and then transfected with miR-122-5p mimic; in the Model+NC group, HEI-OC1 cells were exposed to 50 µM H 2 O 2 for 1 h and then transfected with the blank vector lentivirus; in the Model+FOXO3 group: HEI-OC1 cells were exposed to 50 µM H 2 O 2 for 1 h and then transfected with the FOXO3 lentivirus; in the Model+M+FOXO3 group: HEI-OC1 cells were exposed to 50 µM H 2 O 2 for 1 h and then co-transfected with the miR-122-5p mimic and FOXO3 lentivirus.
Dual luciferase reporter assays. TargetScan V7.2 (www. targetscan.org/vert_72/) was used to explore the targets of miR-122-5p. HEI-OC1 cells (1x10 5 ) were seeded to a 24-well plate and then incubated overnight. Next, pcDNA3. Subsequently, 100 µl transfected cells were placed in a 96-well plate and then added together with 100 µl Dual-Lumi™ firefly luciferase assay reagent or 100 µl Dual-Lumi™ Renilla luciferase assay working solution that was derived from the dual luciferase reporter gene assay kit (RG088M, Beyotime Institute of Biotechnology). After being mixed well, the cells were incubated at room temperature (~25˚C) for 10 min. Finally, cell luciferase was determined with the dual luciferase reporter assay system (Promega Corp.). In this research, dual luciferase reporter assays were performed three times.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR). After being exposed to 50 µM H 2 O 2 for 1 h post transfection, HEI-OC1 cells were collected for RNA extraction using the RNAeasy kit (R0027; Beyotime Institute of Biotechnology) and the miRNA was extracted by the RNAeasy kit (R0028; Beyotime Institute of Biotechnology). RNA was detected using a UV spectrophotometer (DR6000; Hash) and then reversed by the reverse transcription kit (D7168L; Beyotime Institute of Biotechnology) for cDNA synthesis. Finally, cDNA, as a template, was amplified using a real-time fluorescence quantitative PCR instrument (ABI 7500; Thermo Fisher Scientific, Inc.). The conditions of amplification are listed as follows: pre-denaturation at 95˚C for 10 sec, followed by 30 cycles of denaturation at 95˚C for 5 sec and 60˚C for 25 sec, and an elongation at 70˚C for 30 min. The forward and reverse primers for the miR-122-5p sequence, according to Primer3Plus (http://www.primer3plus.com/cgi-bin/dev/prim-er3plus.cgi), were 5'-TGT GAC AAT GGT GTT TGG TCG-3' and 5'-TGT CGT GGA GTC GGC AAT TG-3'; FOXO3 forward primer was 5'-TCA CGC ACC AAT TCT AAC GC-3', and the universal primer was 5'-CAC GGC TTG CTT ACT GAA GG-3'. U6 (forward primer 5'-CTC GCT TCG GCA GCA CA-3' and reverse primer 5'-AAC GCT TCA CGA ATT TGC GT-3') was used as the reference gene, and the 2 -ΔΔCq method was utilized to calculate the expression level (23).
Flow cytometry. Firstly, the cells were resuspended in 400 µl of binding buffer (1X) at a concentration of 1x10 6 cells/ml. After the addition of 5 µl Annexin V-FITC (APOAF-20TST; Sigma-Adrich; Merck KGaA), the cells were cultivated at room temperature for 15 min in the dark, followed by continuous incubation with 10 µl of propidium iodide (PI; P4170; Sigma-Adrich; Merck KGaA) for 5 min. Finally, the fluorescence intensity of cells in each group was measured by flow cytometry (Fortessa X-20; Bio-Rad Laboratories, Inc. USA).
Enzyme-linked immunosorbent assay (ELISA). Diluted cell samples (100 µl) were transferred to a 96-well plate and incubated at room temperature for 2.5 h. After being rinsed 4 times with 1X washing buffer, each well of the plate was added together with 100 µl of prepared biotin conjugate, followed by 1 h of incubation at room temperature with gentle shaking. After rinsing 4 times with 1X washing buffer, the plate was supplemented with 100 µl of the prepared streptavidin-HRP solution, and incubated at room temperature for 45 min with gentle shaking. Following that, the solution was discarded, the plate was rinsed with 1X washing buffer for another 4 times, and each well was added with 100 µl of TMB substrate. Subsequently, the plate was incubated at room temperature for 30 min in the dark with gentle shaking. Afterwards, 50 µl of stop solution was placed into each well, and the side of the plate was tapped to mix the solution well. Finally, 200 µl of supernatant was collected and then added to the 96-well plate, subsequent to which the absorbance was measured at 532 nm using a microplate reader (Z742711-1EA; Sigma-Adrich; Merck KGaA). Lipid peroxidation assay kit (MDA; A0031-2) and superoxide dismutase assay kit (SOD; A001-3-2) applied in the whole processes were purchased from Nanjing Jiancheng Bioengineering Institute.
Confocal laser scanning microscopy analysis. After exposure to 50 µM H 2 O 2 for 1 h, the HEI-OC1 cells were cultured using the ROS Assay Kit (S0033S; Beyotime Institute of Biotechnology), added together with an appropriate volume of diluted 2' ,7'-dichlorodihydrofluorescein diacetate (DCFH-DA; 1:1,000), and then inoculated to a 6-well plate. Then cells were separately dyed by Mito-SOX Red (C1049-50 µg; Beyotime Institute of Biotechnology) staining with a final concentration of 4 µmol/l at 37˚C for 10 min in the dark or CM-H 2 DCFDA (S0033S; Beyotime Institute of Biotechnology) staining at a final concentration of 5 µmol/l at 37˚C for 30 min in the dark. After washing three times with PBS (C0221A; Beyotime Institute of Biotechnology), the cells were captured by laser confocal microscope (LSM800; Zeiss) at different excitation wavelengths (510 nm/488 nm) and different emission wavelengths (580/515 nm), and fluorescence images were captured.
Flow cytometry to detect the mitochondrial membrane potential level. The mitochondrial membrane potential level was measured with the mitochondrial membrane potential assay kit (JC-1; C2006; Beyotime Institute of Biotechnology). Concretely, the cells were collected and then seeded to each cell of a 6-well plate, with the culture medium aspirated. Following being washed once with PBS (C0221A; Beyotime Institute of Biotechnology), the cells were added together with 1 ml of fresh cell culture medium which contained serum and phenol red. Subsequently, 1 ml JC-1 staining working solution was added and mixed well to cultivate the cells in an incubator at 37˚C for 20 min. During the incubation period, an appropriate amount of JC-1 staining buffer (1X) was prepared according to the ratio of 4 ml distilled water per 1 ml JC-1 staining buffer (5X), and placed in an ice bath. After incubation at 37˚C, the supernatant was aspirated and the cells were washed twice with JC-1 staining buffer (1X). Finally, the cells were supplemented with 2 ml of cell culture medium containing serum and phenol red and observed using a flow cytometer (Fortessa X-20; Bio-Rad Laboratories, Inc.).
Statistical analyses. SPSS version 17.0 (SPSS Inc.) was adopted to analyze the statistical data from this research, and these data are represented as the mean ± standard deviation (SD). The Student's t-test or one way analysis of variance (one-way ANOVA) with post hoc Tukey test was utilized to gauge the results for two or multiple groups. P<0.05 was considered as indicative of a statistically significant difference.

miR-122-5p promotes the viability but inhibits the apoptosis of H 2 O 2 -induced HEI-OC1 cells.
HEI-OC1 cells were exposed to 50 µM H 2 O 2 for 1 h (Model group), and subsequently, the expression level of miR-122-5p was observed to be significantly decreased relative to that of the Blank group (Fig. 1A, P<0.001). As shown in Fig. 1B, miR-122-5p mimic (M) transfection significantly increased the level of miR-122-5p in the HEI-OC1 cells (P<0.001). Compared with that of the Blank group, the cell viability of the Model group showed a significant decrease at 24, 48 and 72 h (Fig. 1C, P<0.05). In comparison with that of the Model+mimic control (MC) group, the cell viability of the Model+M group was significantly elevated at 24, 48 and 72 h (Fig. 1C, P<0.05). Additionally, the Model group exhibited a significant increase in the apoptosis rate as compared with Blank group, while the Model+M group had a significantly lower apoptosis rate than the Model+MC group (Fig. 1D, P<0.001). These findings indicated that oxidative stress reduced the cell viability but promoted cell apoptosis. Moreover, high expression of miR-122-5p reversed the effects of the oxidative stress on the viability and apoptosis of the HEI-OC1 cells.
High expression of miR-122-5p partially offsets the effect of H 2 O 2 on the expression levels of apoptosis-related proteins in HEI-OC1 cells. The results revealed that compared with the Blank group, the protein expression levels of Bax, cleaved (C)-caspase-3, and C-caspase-9 in the Model group were significantly upregulated ( Fig. 2A, C-E, P<0.01) as well as the RNA level of Bax (Fig. 2G, P<0.01), while that of Bcl-2 was downregulated at both the protein ( Fig. 2A and B, P<0.001) and RNA level (Fig. 2F, P<0.001). Compared with the Model+MC group, the expression levels of Bax, C-caspase-3, and C-caspase-9 were significantly downregulated ( Fig. 2A, C-E and G, P<0.05), while the Bcl-2 level was significantly upregulated in the Model+M group ( Fig. 2A and B, P<0.001) and also at the RNA level (Fig. 2F, P<0.001). These findings demonstrated that oxidative stress regulated the expression levels of apoptosis-related proteins (Bax, C-caspase-3, C-caspase-9 and Bcl-2), and high expression of miR-122-5p can partially counteract the trend.
High expression of miR-122-5p reduces the levels of ROS and MDA and mitochondrial depolarization, but increases the SOD level under H 2 O 2 condition. The MDA content was significantly elevated (Fig. 3A, P<0.001) while the SOD content was significantly reduced in the Model group (Fig. 3B, P<0.01) as compared with these findings in the Blank group. However, the MDA content was significantly decreased (Fig. 3A, P<0.01) while the SOD content was significantly increased in the Model+M group (Fig. 3B, P<0.05), relative to the Model+MC group. According to DCFH-DA fluorescence detection of the ROS level in the HEI-OC1 cells, compared with the Blank group, the ROS level was significantly increased in the Model group ( Fig. 3C and D, P<0.001); while compared to the Model + MC group, the ROS level was significantly reduced in the Model+M group ( Fig. 3C and D, P<0.01). Mitochondrial membrane potential analysis indicated that the Model group had an significantly increased λ530/λ590 ratio relative to Blank group (P<0.001), while the Model+M group exhibited a statistically decreased λ530/λ590 ratio (P<0.001), when compared with that of the Model+MC group (Fig. 3E). The results above suggest that the high expression of miR-122-5p can partially reverse the oxidative damage of HEI-OC1 cells.

FOXO3 overexpression reverses the effects of miR-122-5p mimic on viability and apoptosis of H 2 O 2 -induced HEI-OC1
cells. WB and RT-qPCR assays demonstrated that the FOXO3 expression level was significantly increased in the Model group ( Fig. 5A-C, P<0.05) after HEI-OC1 cells were treated with H 2 O 2 , compared with that in the Blank group. HEI-OC1 cell viability was determined by MTT assay, as depicted in Fig. 5D. At 24, 48, and 72 h after HEI-OC1 cells were transfected, the cell viability in the Model group was significantly lower than that of the Blank group (P<0.05); the cell viability in the Model+M group was significantly increased as compared with Model+MC group (P<0.001); the cell viability in the Model+FOXO3 group was significantly decreased compared with the Model+NC group (P<0.01), which was offset by miR-122-5p mimic (P<0.01). Flow cytometry was utilized to evaluate the apoptosis rate, as delineated in Fig. 5E. In the Model group, there was a significant increase in the apoptosis rate in comparison with the Blank group (P<0.001). In addition, the Model+M group exhibited a significant decrease in the apoptosis rate (P<0.01) as compared with Model+MC group. And compared with the Model+NC group, the apoptosis rate of cells in the Model+FOXO3 group was significantly increased (P<0.01). Moreover, the apoptosis rate of cells of the Model+M+FOXO3 group was notably lower than that in the Model+FOXO3 group (P<0.01). Moreover, in comparison with the Model+M group, the apoptosis rate of cells in the Model+M+FOXO3 group was dramatically elevated (P<0.01).   (Fig. 6A and B) and mRNA level (Fig. 6F), P<0.001], while the protein levels of Bax (and at the mRNA level as shown in Fig. 6G, P<0.01), C-caspase-3, and C-caspase-9 were significantly elevated in the Model group (Fig. 6A, C-E, P<0.001). As compared with Model+MC group, the expression levels of Bax (at the protein and mRNA levels), C-caspase-3 and C-caspase-9 in the Model+M group were significantly downregulated (Fig. 6A, C-E and G, P<0.01), whereas that of Bcl-2 was significantly upregulated [at the protein level as shown in Fig. A and B, P<0.001; at the mRNA level as shown in Fig. 6F, P<0.001). Relative to the Model+NC group, the level of Bcl-2 (at the protein level as shown in Fig. 6A and B, P<0.05; at the mRNA level as shown in Fig. 6F, P<0.05) was significantly decreased, while levels of Bax (at both the protein and mRNA levels), C-caspase-3, C-caspase-9 were significantly elevated in the Model+FOXO3 group (Fig. 6A, C-E and G, P<0.01). In contrast with the Model+FOXO3 group, the level of Bcl-2 was increased (both at the protein as shown in Fig. A and B; and the mRNA level as shown in Fig. 6F, P<0.05), while levels of Bax (at both the protein and mRNA level), C-caspase-3, and C-caspase-9 were significantly decreased in the Model+M+FOXO3 group (Fig. 6A, C-E and G, P<0.01). These data above indicated that FOXO3 overexpression counteracted the effects of miR-122-5p mimic on the expression levels of apoptosis-related proteins in H 2 O 2 -induced HEI-OC1 cells.

FOXO3 overexpression overturns the effect of miR-122-5p mimic on the levels of ROS, MDA, and SOD, and mitochondrial membrane depolarization in H 2 O 2 -induced HEI-OC1 cells.
As compared with the Model+NC group, the levels of MDA (Fig. 7A), ROS (Fig. 7D) and λ530/λ590 ratio (Fig. 7E) were significantly increased in the Model+FOXO3 group (P<0.05), while the level of SOD (Fig. 7B) was significantly depleted (Fig. 7B, P<0.05). In comparison with the Model+FOXO3 group, the levels of MDA, ROS, together with λ530/λ590 ratio were significantly reduced in the Model+M+FOXO3 group (P<0.01), whereas the level of SOD was significantly elevated (Fig. 7A-D, P<0.01). These data suggested that FOXO3 overexpression neutralized the effects of miR-122-5p mimic on the levels of ROS, MDA, and SOD, and mitochondrial depolarization in H 2 O 2 -induced HEI-OC1 cells.

Discussion
In order to elucidate the regulatory mechanism of miR-122-5p on cochlear hair cells under oxidative stress, we established an oxidative stress model by exposing HEI-OC1 cells to 50 µM H 2 O 2 for 1 h. The results of the present study were utilized to analyze the underlying role of miR-122-5p as a potential target for hearing loss treatment.
Under oxidative stress, reactive oxygen species (ROS) is a well-documented factor in noise-induced hearing loss. In several prior research studies, noise was found to activate AMPKα in outer hair cells (OHCs) through formation of ROS, and noise exposure-induced OHC death was mediated by a ROS/AMPKα-dependent pathway (24)(25)(26). High level of ROS  is associated with hearing loss and hair cell death (2,(27)(28)(29), which can cause changes in the expression levels of related proteins (30). Excess ROS overwhelms the redox balance and skews cell metabolism toward the activation of intrinsic apoptosis, which are regulated by the combined actions of proand anti-apoptotic members of the Bcl-2 family (31)(32)(33)(34). It has been well established that the anti-apoptotic protein Bcl-2 can prevent the release of cytochrome c and reduce the activation of caspase-9 and caspase-3, thus inhibiting caspase-3-dependent apoptosis (30,35). Thus, the degree of oxidative stress can be identified in cells by assessing the expression levels of apoptotic proteins. Apoptosis, MDA production, SOD expression and changes in mitochondrial membrane potential can all be exploited to assess oxidation reactions (36,37). The results in this study signified that under oxidative stress, the cell viability was weakened and apoptosis was enhanced. With regard to the expression levels of apoptosis-related proteins, the Bcl-2 level was decreased, while those of Bax, cleaved (C)-caspase-3, and C-caspase-9 were elevated in HEI-OC1 cells. Additionally, the levels of ROS, MDA and the mitochondrial membrane potential were increased, yet the SOD level was reduced in the HEI-OC1 cells. Seminal miRNA-122 has been manifested to be negatively correlated with oxidative stress, and apoptotic markers (Bax, Bcl-2) in infertile men with varicocele (38). In the present study, the miR-122-5p level was found to be decreased in H 2 O 2 -induced HEI-OC1 cells, and miR-122-5p mimic was able to partially offset the effect of H 2 O 2 on the cell viability and apoptosis, mitochondrial membrane potential levels, as well as apoptosis-and oxidative-related molecules in the HEI-OC1 cells. Taken together, miR-122-5p can attenuate H 2 O 2 -induced oxidative damage in HEI-OC1 cells.
In order to elucidate the possible mechanism of miR-122-5p on cochlear hair cells under oxidative stress, we gained access to the TargetScan V7.2 website to predict the targeting relationship between miR-122-5p and FOXO3. Forkhead box O3 (FOXO3) belongs to the forkhead box O (FOX) family (FKHRL1) that has a common structural motif, namely the 'forkhead box' or 'winged helix' domain that is responsible for binding to chromatin DNA in the nucleus of cells (39). FOXO proteins act as nuclear transcription factors that mediate the inhibitory action of insulin or insulin-like growth factor (IGF-1) on key functions in diverse pathways including cell metabolism, proliferation, differentiation, oxidative stress, cell survival and senescence, autophagy and aging in mammals (39). FOXO3 has important significance in the process of oxidative stress. In the process of self-eating due to oxidative stress, cytoplasmic STAT3 constitutively inhibits autophagy by sequestering EIF2AK2 as well as by  interacting with other autophagy-related signaling molecules such as FOXO1 and FOXO3 (40). Increasing evidence demonstrates that multiple miRNAs can also synergistically act on FOXO3a, thus playing important roles in the development and progression of various human diseases (41)(42)(43). Additionally, overexpression of miR-182 represses the intrinsic apoptotic pathway by inhibiting the translation of FOXO3a, protecting cochlear hair cells from cisplatin-induced apoptosis in the inner ear (44). Gentamicin-induced cochlear hair cell ototoxicity, including oxidative stress and apoptosis, could be attenuated by mouse inner ear stem cells (IESCs) through the miR-182-5p/FOXO3 axis (45).
In the present study, it was found that the FOXO3 level was increased in H 2 O 2 -induced HEI-OC1 cells, and FOXO3 overexpression could further promote the effects of H 2 O 2 on the viability and apoptosis, mitochondrial membrane potential levels, as well as apoptosis-and oxidative-related molecules in HEI-OC1 cells. In addition, the present findings also indicated that FOXO3 overexpression can partially offset the effect of high expression of miR-122-5p in H 2 O 2 -induced HEI-OC1 cells. Despite these achievements, our research still had some shortcomings. Only in vitro experiments, no in vivo experiments were conducted. The research also lacked a morphological basis. These should be explored in the further study. The above results illustrated that miR-122-5p can attenuate the H 2 O 2 -induced damage in mouse cochlear hair cells by targeting FOXO3.
In conclusion, the oxidative stress damage of hair cells caused by H 2 O 2 can be alleviated by inhibiting the expression of FOXO3 or promoting the expression of miR-122-5p, providing a new perspective and scientific basis for the effective treatment of hearing impairment or loss.