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.
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 (
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 (
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 H2O2-induced damage in mouse cochlear hair cells by directly regulating FOXO3.
The House Ear Institute-Organ of Corti 1 (HEI-OC1) cell line was obtained from the Medical Experimental Center of Guangzhou Red Cross Hospital (China). High-glucose Dulbecco's modified Eagle's medium (DMEM) (30-2002, American Type Culture Collection, Beijing, China) supplemented with 10% fetal bovine serum (FBS; C0257, Beyotime Institute of Biotechnology) was used to culture the HEI-OC1 cells at 33˚C in a humidified incubator with 5% CO2.
The miR-122-5p mimic (M; 5'-UGGAGUGACAAUGGUGUUUG-3'), mimic control (MC; 5'-UUCUCCGAACGUGUCACGUTT-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 1x105 cells/well. The density of the cells during lentiviral transfection was ~2x105 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).
HEI-OC1 cells were exposed to 50 µM hydrogen peroxide (H2O2) for 1 h post transfection to mimic oxidative stress condition, as previously reported (
TargetScan V7.2 (
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™
After being exposed to 50 µM H2O2 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 (
Cells (1x106-1x107) were taken from each group as samples, and then washed with phosphate-buffered saline (PBS; C0221A; Beyotime Institute of Biotechnology). Next, the cell samples were added with 0.5 ml total protein extraction reagent to extract the total protein. Based on the instructions of the total protein extraction kit (W034-1-1; Nanjing Jiancheng Bioengineering Institute,
After being transfected, cells (2x103 cells/well) were seeded into a 96-well plate and maintained at 37˚C in 100 µl of culture medium. After transfection for 24, 48, 72 and 96 h, the cells in each well were supplemented with 3-(
Firstly, the cells were resuspended in 400 µl of binding buffer (1X) at a concentration of 1x106 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).
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.
After exposure to 50 µM H2O2 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-H2DCFDA (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.
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.).
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.
HEI-OC1 cells were exposed to 50 µM H2O2 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 (
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 (
The MDA content was significantly elevated (
TargetScan V7.2 (
WB and RT-qPCR assays demonstrated that the FOXO3 expression level was significantly increased in the Model group (
Compared to the Blank group, the level of Bcl-2 was significantly decreased [at both the protein (
As compared with the Model+NC group, the levels of MDA (
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 H2O2 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 (
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 (
In the present study, it was found that the FOXO3 level was increased in H2O2-induced HEI-OC1 cells, and FOXO3 overexpression could further promote the effects of H2O2 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 H2O2-induced HEI-OC1 cells. Despite these achievements, our research still had some shortcomings. Only
In conclusion, the oxidative stress damage of hair cells caused by H2O2 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.
Not applicable.
The analyzed datasets generated during the study are available from the corresponding author on reasonable request.
JC made substantial contributions to conception and design of the study. JQ and JL were responsible for the data acquisition, data analysis and interpretation and confirm the authenticity of all the raw data. JJ performed the drafting of the article and critically revised it for important intellectual content. All authors read and approved the final manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved.
Not applicable.
Not applicable.
The authors declare no competing interests.
miR-122-5p increases the viability but inhibits the apoptosis of hydrogen peroxide (H2O2)-exposed HEI-OC1 cells. (A) Expression level of miR-122-5p in HEI-OC1 cells after oxidative damage of H2O2 (Model) was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). ***P<0.001 vs. the Blank group. (B) Expression level of miR-122-5p in HEI-OC1 cells after transfection with the miR-122-5p mimic (M) or mimic control (MC) was detected by RT-qPCR. ^^^P<0.001 vs. the MC group. (C) Viability of the HEI-OC1 cells following oxidative damage of H2O2 (Model) was determined by MTT assay in the different groups. #P<0.05 and ###P<0.001 vs. the Model+MC group. *P<0.05 and ***P<0.001 vs. the Blank group. (D) Apoptosis of HEI-OC1 cells after oxidative damage of H2O2 was measured by flow cytometry in the different groups. ***P<0.001 vs. the Blank group; ###P<0.001 vs. the Model + MC.
High expression of miR-122-5p can partially offset the effect of H2O2 on the expression levels of apoptotic molecules in HEI-OC1 cells. (A) Levels of Bcl-2, Bax, C-caspase-3 and C-caspase-9 in H2O2-induced HEI-OC1 cells were determined by western blot (WB) analysis. (B) Expression level of Bcl-2 in H2O2-induced HEI-OC1 cells was quantified by WB analysis. ***P<0.001 vs. the Blank group; ###P<0.001 vs. the Model+MC group. (C) Expression level of Bax in HEI-OC1 cells after oxidative damage of H2O2 was measured by WB analysis. **P<0.01 vs. the Blank group. #P<0.05 vs. the Model+MC group. (D) Expression level of C-caspase-3 in H2O2-induced HEI-OC1 cells was tested by WB analysis. ***P<0.001 vs. the Blank group. ##P<0.01 vs. the Model+MC group. (E) Expression level of C-caspase-9 in H2O2-induced HEI-OC1 cells was detected by WB analysis. **P<0.01 vs. the Blank group. ##P<0.01 vs. the Model+MC group. (F) mRNA expression level of Bcl-2 in H2O2-induced HEI-OC1 cells was quantified by RT-qPCR. ***P<0.001 vs. the Blank group. ###P<0.001 vs. the Model+MC group. (G) mRNA expression level of Bax in H2O2-induced HEI-OC1 cells was determined by RT-qPCR. **P<0.01 vs. the Blank group. ##P<0.01 vs. the Model+MC group. Model, cells exposed to 50 µM H2O2 for 1 h; M, miR-122-5p mimic; MC, mimic control; C-, cleaved.
High expression of miR-122-5p reduces the levels of ROS and MDA and mitochondrial depolarization, but increases the SOD level under H2O2 condition. (A) MDA content in H2O2-induced HEI-OC1 cells was tested by enzyme-linked immunosorbent assay (ELISA). ***P<0.001 vs. the Blank group. ##P<0.01 vs. the Model+MC group. (B) SOD content in H2O2-induced HEI-OC1 cells was evaluated by ELISA. **P<0.01 vs. the Blank group. #P<0.05 vs. the Model+MC group. (C and D) ROS content in H2O2-induced HEI-OC1 cells was determined by laser scanning confocal microscope. ***P<0.001 vs. the Blank group. ##P<0.01 vs. the Model+MC group. (E) Cell membrane potential level in H2O2-induced HEI-OC1 cells was detected by flow cytometry. ***P<0.001 vs. the Blank group. ###P<0.001 vs. the Model+MC group. Model, cells exposed to 50 µM H2O2 for 1 h; M, miR-122-5p mimic; MC, mimic control.
miR-122-5p directly targets FOXO3. (A) The 3'UTR sequences of FOXO3-wild-type (WT), FOXO3-mutant (MUT) and miR-122-5p. (B) Dual luciferase reporter assay was applied to analyze the fluorescence activity in H2O2-induced HEI-OC1 cells. ***P<0.001. vs. MC. FOXO3, forkhead box O3; M, miR-122-5p mimic; MC, mimic control.
FOXO3 overexpression offsets the effects of miR-122-5p mimic (M) on viability and apoptosis of H2O2-induced HEI-OC1 cells. (A) Expression level of FOXO3 in H2O2-induced HEI-OC1 cells (Model) was measured by western blot analysis. (B) The protein expression level of FOXO3 in H2O2-induced HEI-OC1 cells was quantified by western blot analysis. *P<0.05 vs. the Blank group. (C) The mRNA expression level of FOXO3 in H2O2-induced HEI-OC1 cells was determined by RT-qPCR. ***P<0.001 vs. the Blank group. (D) Viability of the H2O2-induced HEI-OC1 cells in the different groups was evaluated by MTT assay. (E) Apoptosis of H2O2-induced HEI-OC1 cells was analyzed by flow cytometry. **P<0.01 and ***P<0.001 vs. the Blank group; #P<0.05, ##P<0.01 and ###P<0.001 vs. the Model+MC group; ^P<0.05, ^^P<0.01 and ^^^P<0.001 vs. the Model+M group; §§P<0.01 vs. the Model+NC group; ‡‡P<0.01 and ‡‡‡P<0.001 vs. the Model + FOXO3. Model, cells exposed to 50 µM H2O2 for 1 h; M, miR-122-5p mimic; MC, mimic control; NC, negative control; FOXO3, forkhead box O3.
FOXO3 overexpression reverses the effect of miR-122-5p mimic on the expression levels of apoptosis-related proteins in H2O2-induced HEI-OC1 cells. (A) HEI-OC1 cells were transfected with miR-122-5p mimic (M), mimic control (MC), FOXO3 overexpression vector or negative control (NC), and the expression levels of Bcl-2, Bax, cleaved-caspase-3 and C-caspase-9 were detected by western blot (WB) analysis in HEI-OC1 cells after oxidative damage of H2O2. (B) The protein expression level of Bcl-2 was quantified by WB analysis in HEI-OC1 cells after oxidative damage of H2O2. (C) The protein expression level of Bax was measured by WB analysis in HEI-OC1 cells after oxidative damage of H2O2. (D) The protein expression level of C-caspase-3 in H2O2-induced HEI-OC1 cells was tested by WB analysis. (E) The expression level of C-caspase-9 in H2O2-induced HEI-OC1 cells was assessed by WB analysis. (F) The mRNA expression level of Bcl-2 in H2O2-induced HEI-OC1 cells was quantified by RT-qPCR. (G) The mRNA expression level of Bax in H2O2-induced HEI-OC1 cells was determined by RT-qPCR. **P<0.01 and ***P<0.001 vs. the Blank group; ##P<0.01 and ###P<0.001 vs. the Model+MC group; ^^P<0.01 and ^^^P<0.001 vs. the Model+M group; §P<0.05, §§P<0.01 and and §§§P<0.001 vs. the Model+NC group; ‡P<0.05, ‡‡P<0.01 and ‡‡‡P<0.001 vs. the Model + FOXO3. Model, cells exposed to 50 µM H2O2 for 1 h; M, miR-122-5p mimic; MC, mimic control; NC, negative control; FOXO3, forkhead box O3 overexpression vector; C-, cleaved.
FOXO3 overexpression reverses the effects of miR-122-5p mimic on the levels of ROS, MDA, and SOD, and mitochondrial depolarization in H2O2-induced HEI-OC1 cells. (A) The content of MDA in transfected HEI-OC1 cells after oxidative damage of H2O2 was assessed by ELISA. (B) The content of SOD in transfected HEI-OC1 cells after oxidative damage of H2O2 was evaluated by ELISA. (C and D) ROS content of transfected HEI-OC1 cells after oxidative damage of H2O2 was detected by laser scanning confocal microscopy. (E) Cell membrane potential level of transfected HEI-OC1 cells after oxidative damage of H2O2 was determined by flow cytometry. **P<0.01 and ***P<0.001 vs. the Blank group; #P<0.05, ##P<0.01 and ###P<0.001 vs. the Model+MC group; ^P<0.05, ^^P<0.01 and ^^^P<0.001 vs. the Model+M group; §P<0.05 and §§P<0.01 vs. the Model+NC group; ‡‡P<0.01 vs. the Model + FOXO3. Model, cells exposed to 50 µM H2O2 for 1 h; M, miR-122-5p mimic; MC, mimic control; NC, negative control; FOXO3, forkhead box O3 overexpression vector.