Inhibition of KDM5A attenuates cisplatin-induced hearing loss via regulation of the MAPK/AKT pathway

Compounds

CPI-455 (S8287; Selleckchem, Houston, TX, USA) was dissolved as 10 mmol/L of stock solution with DMSO (D5879; Sigma-Aldrich, St. Louis, MO, USA). CP was purchased from Sigma-Aldrich (479,306; Sigma-Aldrich) and was initially dissolved in PBS at a stock concentration of 3 mmol/L and further diluted in the medium used for cell and explant culture analysis.

Cell cultures

HEI-OC1 cells were cultured in high-glucose DMEM (11965-092; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (10099-141; Gibco) at 33 °C under a 10% CO2 condition [73,74,75,76,77]. FaDu and HGC-27 cell lines, preserved in our laboratory, were used before the 15th passage, and all experiments were performed in triplicate. Both cells were cultured in RPMI-1640 medium (C11875500BT; Gibco) containing 10% FBS and penicillin–streptomycin (15140-122; Gibco) under a 5% CO2 condition. The cells were trypsinized with 0.25% trypsin/EDTA (25200056; Gibco) at 80% confluence.

Postnatal cochlear and SGN explants cultures and drug treatments

Animal experiments and research protocols were in accordance with the Fudan University Institutional Animal Care and Use Committee. Cochlea or SGNs explants from C57BL/6 mice at postnatal day 2 were dissected in PBS and attached to cell-Tak-coated coverslips. Then, explants were cultured in DMEM/F12 (C11330500BT; Gibco), containing 1% N2 (100 ×, 17502-048; Gibco) supplement, 2% B27 (50 ×, 17504-044; Gibco) supplement, and ampicillin (P0781; Sigma-Aldrich) at 37 °C and 5% CO2.

In vivo study

Adult 8-week-old C57BL/6 mice were randomly divided into the following groups: control, CP, CPI-455, and CPI-455 + CP groups. 1 mg/mL CP stock solution was prepared in sterile saline (0.9% NaCl). The mice in the CP group received three rounds of once-daily intraperitoneal injections of CP at a dose of 3.5 mg/kg for 4 days, followed by a 10-day recovery period (a total of 42 days), as previously described [23, 78]. Mice in the CPI-455 + CP group received intraperitoneal injections of CPI-455 (0.5 mg/kg or 2 mg/kg) starting 1 day before the injection of CP, and each cycle lasted for 5 days. Mice in the control and CPI-455 groups were injected with the same volume of saline or CPI-455, respectively.

For the high-dose CP-induced ototoxicity experiment, C57BL/6 mice were injected intraperitoneally with 1 mL of warm saline. After 24 h, CPI-455 was injected intraperitoneally 2 h in advance in mice of the CPI-455 + CP group and CPI-455 group; then, a single dose of CP (30 mg/kg) was injected intraperitoneally in mice of the CPI-455 + CP group and CP group. The ABR threshold was measured 14 days after administration.

ABR

ABR recording was implemented with the TDT system (Tucker Davis Technologies, Alachua, FL, USA). Mice were commonly anesthetized using a ketamine (100 mg/kg)–xylazine (10 mg/kg) cocktail before measuring ABR. The recording, reference, and ground electrodes were inserted at the midpoint of the line joining the anterior edge of the auricle on each side, behind the tested ear, and behind the contralateral ear of the mouse. The SigGenRZ software (Tucker Davis Technologies) performed the ABR tests at 8-, 16-, 24-, and 32-kHz frequencies. The tone bursts were selected to evoke ABR, and the sound intensity was attenuated gradually from 90 to 20 dB at intervals of 5 dB. The response for each frequency level was analyzed, digitized, and averaged using the BioSigRZ software (1024 samples/level). The amplitude of ABR wave I was determined by analyzing ABR waveforms.

ROS detection

ROS production was detected using CellROX™ green (C10444; Invitrogen, Carlsbad, CA, USA) and MitoSOX™ red (M36008; Invitrogen) according to the method described previously [79, 80]. For analyzing ROS levels in HEI-OC1 cells, cells were incubated with 5 μmol/L of CellROX™ green (excitation/emission maxima of 488/520 nm) working solution at 37 °C for 30 min in the dark. Then, HEI-OC1 cells were stained with 10 µg/mL Hoechst 33,258 (excitation/emission maxima of 350/461 nm) (861,405; Sigma–Aldrich) for 5 min, and the images were observed and captured with a confocal fluorescence microscope (Leica TCS SP8; Leica Microsystems GmbH, Wetzlar, Germany). Fluorescent signal intensity was taken with flow cytometry. Flow cytometric analysis used a Beckman Coulter MoFlo XDP (Miami, FL, USA) with FlowJo software (Version 10; TreeStar, Ashland, OR). CellROX™ fluorescence intensity was detected at an excitation wavelength of 488 nm and an emission wavelength of 520 nm (FL-1) by flow cytometry with gating at 20,000 cells/sample and the mean fluorescence intensity used to represent the amount of ROS. All experiments were repeated three times.

Regarding ROS assessment in the cochlea, cochlear explants from different treatments were incubated with 5 μmol/L of MitoSOX™ red (excitation/emission maxima of 510/580 nm) at 37 °C for 30 min in the dark. Fluorescent signal intensity was taken with fluorescence microscopy. Briefly, the representative images of the cochleae in each turn were randomly captured with a confocal fluorescence microscope under the same settings. MitoSOX™ red immunofluorescence was quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA) from raw confocal images that were taken under the same conditions at 63 × magnification lens with a 0.75 × digital zoom and with the same laser gain and PMT gain parameters. Cochlear explants were stained with a rabbit polyclonal antibody to Myosin 7a (1:500, 25–6790; Proteus Biosciences, Ramona, CA, USA) to determine comparable portions of the HCs in confocal images. Grayscale values were determined in the HCs to quantify changes in fluorescence intensity. The MitoSOX™ red signal was measured in the apical, middle, and basal turns of cochlear explant. The intensity of the background was subtracted and the mean intensity was then calculated. For each replicate, the relative fluorescence intensity values were determined by ratio normalization with the control group.

MMP measurement

MMP level was measured with Rhodamine 123 (C0062; LifeSct LLC, Rockville, USA) staining [79]. HEI-OC1 cells were incubated with 1 μmol/L of Rhodamine 123 at 37 °C for 30 min. Cells were then visualized under the same settings using a confocal fluorescence microscope.

For analyzing MMP levels in HEI-OC1 cells, cells were treated with trypsin, centrifuged at 1000 rpm for 5 min, and then rinsed twice with PBS. Next, HEI-OC1 cells were incubated in the dark with 1 μmol/L of Rhodamine 123 for 30 min at 37 °C, followed by flow cytometric analysis using the FACScan flow cytometer (Beckman Coulter) for a cell count of 20,000, and the data were analyzed using FlowJo software. Rhodamine 123 fluorescence intensity was detected at an excitation wavelength of 488 nm and an emission wavelength of 534 nm and the mean fluorescence intensity used to represent the levels of MMP. The experiments were repeated three times.

Apoptosis detection

For the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) (11684795910; Roche Holding, Basel, Switzerland) test, cochlear explants were washed with PBS for 15 min and then treated with the reaction mixture at 37 °C for 30 min. Myosin 7a and DAPI were used for HCs and nuclei counterstain, respectively. Representative images of each turn of the cochleae were captured randomly with a confocal fluorescence microscope under the same settings. The number of Myosin 7a/TUNEL-positive cells were counted in the images, and the results were averaged for three images per turn (apex, middle, and base) and expressed as data per 200 µm.

For the Annexin V conjugated to FITC (556547; BD Biosciences, San Jose, CA, USA) assay, HEI-OC1 cells were harvested and washed in PBS, then 2 × 105 cells were resuspended in a solution containing Annexin V (5 μL) and PI (5 μL) and incubated for 20 min. After incubation, 300 μL of binding buffer was added and the cells were analyzed by flow cytometry. All experiments were repeated three times. Cells with positive Annexin V‑FITC staining and negative PI staining were considered to be undergoing early apoptosis, and cells with positive Annexin V‑FITC and PI staining were considered to be undergoing late apoptosis.

Cell viability detection

CCK-8 kit (CK04; Dojindo Laboratories) was used to measure the viability of the cells. Briefly, HEI-OC1 cells were seeded in 96-well plates. After drug treatment, 10 μL of Cell Counting Kit-8 solution and 90 μL of culture medium were mixed, added to each well, and incubated for 3 h. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Seahorse assay

For evaluating the energy metabolism, HEI-OC1 cells were seeded in 180 μL of DMEM containing 10% FBS on Seahorse XF96-cell culture microplates (Seahorse Bioscience, Billerica, MA, USA) at the density of 1.5 × 104 cells/well. Cells were washed with ~ 180 μL of XF assay medium before the assay, then cultured at 37 °C for 60 min. OCR was detected by the Seahorse XF96 instrument (Seahorse Bioscience) following the injections of mixtures in the Seahorse XF Cell Mito Stress Kit (Seahorse Bioscience). The following mixtures were added in proper order: first, oligomycin (complex V inhibitor; 25 µL in port A) at a concentration of 2 μmol/L; second, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 25 µL in port B) at the concentration of 0.5 mmol/L; and, finally, rotenone/antimycin A (25 µL) (inhibitors of complex I and III; in port C) at the concentration of 0.1 μmol/L/1 μmol/L. The XF Cell Mito Stress Test Report Generator software (version 2.6.0.31) was used to calculate and integrate the experiment data, and the results were obtained after triplicate repetition. OCR normalization was defined as total protein/well.

KDM5A-siRNA transfection

HEI-OC1 cells (1 × 103) were seeded in six-well plate and transfected with KDM5A-siRNA at a 50 nmol/L concentration with Lipo 3000 Transfection Reagent (L3000001; Invitrogen) for 24 h. The transfection efficiency of knockdown was detected by qRT-PCR. The following KDM5A-siRNA was used: (1) KDM5A siRNA-01, sense: 5′-GCACAAUCCUAUGACACUUGG-3′, antisense: 5′-CCAAGUGUCAUAGGAUUGUGC-3′. (2) KDM5A siRNA-02, sense: 5′-GCAAAUGAGACAACGGAAA-3′, antisense: 5′-UUUCCGUUGUCUCAUUUGC-3′.

Immunofluorescence

Samples fixed with 4% PFA were permeabilized with 1% PBST for 30 min. Afterward, we blocked with 10% donkey serum in PBST for 1 h, followed by incubation with the following primary antibodies: Myosin 7a rabbit polyclonal antibody (1:500, 25–6790; Proteus Biosciences, Ramona, CA, USA), Tuj-1 mouse (IgG2a) monoclonal antibody (1:1000, 801202; Biolegend, USA), parvalbumin mouse monoclonal antibody (1:1000, ab277625; Abcam, Cambridge, MA, USA), CtBP2 mouse (IgG1) monoclonal antibody (1:1000, 612044; BD Transduction Laboratories, BD Biosciences), GluR2 mouse (IgG2a) monoclonal antibody (1:1000, MAB397; Millipore Corp., Billerica, MA, USA), H3K4me3 rabbit monoclonal antibody (1:500, 9751; Cell Signaling Technology, CST Danvers, MA, USA), and KDM5A rabbit monoclonal antibody (1:500, 3876; CST). Samples were then washed with PBS, followed by incubation with Alexa Fluor 488 donkey anti-rabbit/mouse or cy3-conjugated secondary antibody (1:1000, Invitrogen) along with DAPI (1:2000, D9542; Sigma-Aldrich) for 2 h. For image acquisition, all images were scanned with a laser scanning confocal fluorescence microscope (SP8; Leica) with a 40 × oil immersion objective lens as seen in Fig. 4A–C, or 63 × oil immersion objective lens as seen in Figs. 1A–C, 2A–C, 3A–C, 5D–F, 6A–C, 7C–D, 8D, G, and S1D–F under identical z-stack conditions (in 1 μm Z-steps) and the same laser intensity, gains, and PMT gains parameter settings.

H&E staining

After mice were sacrificed, their hearts, livers, spleens, and lungs were harvested for H&E staining. The sections were deparaffinized twice with xylene (for 20 min each), then rehydrated twice with 100% alcohol (for 5 min each), then rehydrated with 75% alcohol (for 5 min each) and rinsed with water. The sections were stained with Harris hematoxylin solution, counterstained with eosin solution, then dehydrated three times in 100% alcohol (5 min each), and washed twice with xylene (5 min each). Finally, these sections were observed under a light microscope.

Western blot analysis

Protein lysate from cells and cochlear sensory epithelia for each group was prepared in RIPA buffer (P0013B; Beyotime Biotech) with the addition of protease inhibitor (04693132001; Sigma-Aldrich), followed by placement on ice for 30 min, being shaken every 10 min. After the centrifugation at 12,000 rpm at 4 °C for 10 min, the supernatants were denatured and loaded on 12% SDS-PAGE gel. Proteins were then transferred in an ice bath to polyvinylidene difluoride membrane (Immobilon-P, IPVH00010; Millipore, Schaffhausen, Switzerland). After blocking with TBST buffer containing 5% milk for 1 h, the membrane was incubated with primary antibodies overnight at 4 °C, followed by secondary antibody. The antibodies were as follows: cleaved caspase-3 rabbit polyclonal antibody (1:500, 9661; CST), caspase-3 rabbit polyclonal antibody (1:500, 9662; CST), Bax rabbit monoclonal antibody (1:500, 14,796; CST), Bcl-2 rabbit monoclonal antibody (1:500, 3498; CST), phospho-Akt (Ser473) rabbit monoclonal antibody (1:500, 4060; CST), Akt rabbit monoclonal antibody (1:500, 4691; CST), phospho-PI3K rabbit monoclonal antibody (1:500, 17,366; CST), PI3K rabbit monoclonal antibody (1:500, 4257; CST), phospho-p38 MAPK (Thr180/Tyr182) rabbit monoclonal antibody (1:500, 4511; CST), p38 MAPK rabbit polyclonal antibody (1:500, 9212; CST), phospho-JNK rabbit monoclonal antibody (1:1,000, ab124956; Abcam), JNK rabbit monoclonal antibody (1:1,000, ab179461; Abcam), KDM5A rabbit monoclonal antibody (1:500, 3876; CST), KDM5B rabbit polyclonal antibody (1:500, 3273; CST), KDM5C rabbit monoclonal antibody (1:500, 5361; CST), H3K4me3 rabbit monoclonal antibody (1:500, 9751; CST), horseradish peroxidase-linked donkey anti-rabbit IgG polyclonal antibody (1:3,000, 406,401; Biolegend, San Diego, CA, USA), and goat anti-mouse IgG polyclonal antibody (1:3,000, GTX213111-01; GeneTex, Irvine, CA, USA). Each experiment was repeated three times, and GAPDH mouse monoclonal antibody (1:1,000, 437,000; Invitrogen) was used as an internal reference protein. Finally, signals were detected by GE Healthcare’s ECL detection reagent (GE Healthcare, Chicago, IL, USA).

RNA-Seq and real-time RT-PCR

Total RNA integrity was assessed using an Agilent 2100 biological analyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with RNA integrity values of > 7.0 were used for RNA-Seq. The complementary DNA libraries were sequenced on the Illumina HiSeq X Ten platform. The differential expression was analyzed using the software DESeq (2012) R package. P < 0.05, fold-change > 2, and FDR < 0.01 were set as the thresholds for significant differential expression. Based on the hypergeometric distribution, R was used to enrich differentially expressed genes by Gene Ontology and KEGG, respectively. For quantification of gene expression, the 2−ΔΔCT method was used and normalized against GAPDH expression. The primers of all primer sets used in these experiments are shown in Online Table 1.

CUT&Tag

CUT&Tag for bench-top application was performed using 100,000 cells as previously published [81]. More detailed information can be found at the website: http://www.protocols.io/view/bench-top-cut-amp-tag-wnufdew/abstract. The primary antibody was H3K4me3 rabbit monoclonal antibody (1:500, 9751; CST), and the secondary antibody was goat anti-rabbit IgG (AS070; ABclonal, Wuhan, China). The DNAs were extracted and subjected to PCR. The primers of all primer sets used in these experiments are shown in Online Table 1. The experiments were repeated three times independently and the results were normalized to the IgG control.

HC, SGN, and synapse counts

Quantification of HCs, SGNs, and synapses was performed in the apical, middle, and basal turns of the cochlea. For the evaluation of HC, we took 63 × magnified confocal images of each turn of the cochlea at random as representative images. We calculated the number of total HCs in the images, averaged the results for three images from each turn, and presented the data as per 200 µm. For cultured SGN explants, the fluorescence images were acquired using confocal laser microscopy with a 40 × oil immersion objective with a 0.75 × digital zoom and a 1 µm step size between each plane. The length of SGN neurites and the SGN density per 100 μm were calculated. For synaptic quantification, we obtained confocal z-stacks images of IHC area from each turn with a 63 × oil immersion objective with a 2 × digital zoom and a 1 µm step size between each plane. Z-stacks were allowed to span all IHCs, including all synaptic ribbons. We imported the image stacks into ImageJ (National Institutes of Health, Bethesda, MD, USA) and then manually calculated the CtBP2 or GluR2 puncta in each image stack. The number of CtBP2 or GluR2 puncta per IHC within a 100 μm range was calculated in three different regions of every turn of the cochlea.

Statistical analyses

Data were analyzed and plotted by GraphPad Prism software (San Diego, CA, USA) and shown as mean ± SEM. Quantitative data statistical analysis was assessed using one-way analysis of variance (ANOVA) for multiple group comparisons or two-tailed Student's t tests for two-group data. P < 0.05 was considered as significant.

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