HEI-OC1 auditory cell line was a gift from Professor Federico Kalinec (David Geffen School of Medicine at UCLA, CA, USA) and maintained at 32°C in 10% CO₂ in high-glucose Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare Life Sciences) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate and 10% fetal bovine serum (FBS; HyClone).

HEI-OC1 auditory cells were separately treated for 48 h at 32°C in 10% CO₂ with Tan IIA (3, 9, 27, 81, 243 or 729 mg/l; Shanghai Yuanye Biotechnology Co., Ltd.), Tet (125, 250, 500, 1×10³, 2×10³, 4 10³, 8×10³ or 1.60×10⁴ mg/l; Shanghai Aladdin Bio-Chem Technology Co., Ltd.) or cisplatin (5, 10, 20, 40, 80 and 160 µM; Sigma-Aldrich; Merck KGaA). In a separate experiment, the cells were treated for 30 h with 30 µM cisplatin combined either with Tan IIA (0.2, 0.5, 1.0, or 1.5 mg/l) or with Tet (37.5, 75, 125 or 250 mg/l) at 32°C in 10% CO₂. The assays were carried out in triplicate wells.

Cells were seeded into 96-well plates (Corning, Inc.) at a density of 5×10⁴ cells/ml and maintained in high-glucose DMEM for 24 h. The culture medium was then replaced with fresh medium supplemented with Tan IIA, Tet, cisplatin or combinations as aforementioned. Cell viability was then assessed using Cell Counting Kit-8 (MedChemExpress) according to the manufacturer's instructions. The optical density was read at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

Cell apoptosis was measured using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; Beyotime Institute of Biotechnology) staining. Cells (5×10⁴ cells/well) were plated in 6-well plates, then treated in triplicate with cisplatin for 48 h or with cisplatin and Tet for 30 h at 32°C in 10% CO₂. Following incubation, cells were collected by centrifugation at 4°C and 1,500 × g for 5 min, then resuspended in 1X binding buffer. The samples were then stained with 5 µl of Annexin-V-FITC solution and 5 µl of PI at 4°C for 10–15 min in dark. The data were acquired using a BD FACSCanto-II flow cytometer (BD Biosciences) and analyzed using an Olympus BX51 fluorescence microscope (Olympus Optical, Tokyo, Japan).

Proteins were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Protein concentration was determined using a Bradford protein assay kit (Thermo Fisher Scientific Inc.). The samples (20 µg) were resolved by SDS-PAGE on 10% gels (Shanghai Sangong Pharmaceutical Co., Ltd.), then transferred to PVDF membranes (EMD Millipore). The membranes were then blocked with 5% nonfat milk (Beyotime Institute of Biotechnology) at room temperature for 1 h. The membranes were first incubated with target-specific primary antibodies, including anti-P21 (1:1,000; cat. no. ab188224; Abcam), anti-p16-INK4A (1:1,000; cat. no. ab211542; Abcam), anti-FOXO3 (1:1,500; cat. no. 2497S; Cell Signaling Technology, Inc.), anti-caspase 3 (1:1,000; cat. no. ab13847; Abcam), anti-Fas (1:2,000; cat. no. ab216991; Abcam), anti-Wnt receptor frizzled 6 (FZD6; 1:2,000; cat. no. DF4930; Affinity Biosciences), anti-transcriptional repressor transcription factor 7-like 1 (TCF7L1; 1:1,500; cat. no. ab86175; Abcam), anti-wingless-type MMTV integration site family, member 2 (WNT2; 1:800; cat. no. ab109222; Abcam), anti-insulin-like growth factor 1 (IGF1; 1:1,500; cat. no. ab182408; Abcam), anti-SERPINE1 (1:1500; cat. no. DF13553; Affinity Biosciences) and anti-GAPDH (1:10,000; cat. no. KC-5G5; Shanghai Kangcheng Biotechnology Co. Ltd.) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG; 1:20,000; cat. no. ba1054; Boster Biological Technology, Ltd.) at room temperature for 40 min. Lastly, protein bands were visualized using an ECL kit (EMD Millipore).

Total RNA was extracted using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 48 and 30 h post-treatment. RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) and an Agilent 2100 Bioanalyzer (Agilent Technologies Inc.). RNA samples with an RNA integrity number (RIN) value of > eight were used for library preparation. RNA was fragmented, reverse transcribed to the first-strand cDNA using reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) and random hexamers (Sangon Biotech Co., Ltd.), then synthesized to double-stranded DNA using dNTPs (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 15 min and at 98°C for 5 min. The DNA sequencing libraries were prepared using a TruSeq™ RNA Sample Preparation Kit (version 2; Illumina, cat. nos. RS-122-2001 RS-122-2002) following the protocols including DNA fragment end repair, adenylating, adapter ligation, and fragment amplification. After verifying the library quality using the Qubit2.0 (Thermo Fisher Scientific, Inc.), Agilent 2100 (Agilent Technologies Inc.), and qPCR, sequencing (loading concentration 3 nM/µl) was carried out using a V1 sequencing kit (Illumina, Inc.) on an Illumina HiSeq 4000 sequencing platform (paired-end; 150 bp; Illumina, Inc.).

Base calling of the original sequencing image data and fastq file extraction were carried out using Illumina CASAVA software (version 1.8.2; Illumina, Inc.). The quality of raw data was checked using the FastQC program (version 0.11.5; http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). After adaptor trimming and removal of duplicated and low-quality reads (unknown base sequence >10%, and >50% of the read consisted of reads with Phred scores of ≤3) using Trimmomatic (version 0.30; http://www.usadellab.org/cms/?page=trimmomatic), the clean reads were mapped to the ENSEMBL Mus musculus GRCm38.p6 reference genome (http://www.ensembl.org/) with annotation version GRCm38.97 using the TopHat 2 software (http://ccb.jhu.edu/software/tophat/index.shtml).

Transcript assembly and quantification was performed using the Cufflinks software (version 2.2.1; http://cufflinks.cbcb.umd.edu/) by calculating the expected number of fragments per kilobase of exon model per million reads mapped (FPKM) values in each sample. Differentially expressed genes (DEGs) between groups were identified using the edgeR statistical software package (Bioconductor; http://www.bioconductor.org/) according to the criteria of FDR <0.05 and |log₂(FC)|≥1, where FDR is the false discovery rate and FC is the fold change.

Gene Ontology (GO; http://www.geneontology.org/) biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with the DEGs were identified from the Database for Annotation, Visualization, and Integrated Discovery (DAVID; version 6.7; http://david.ncifcrf.gov/). P<0.05 was used to identify statistically significant terms.

The interaction between DEGs was predicted and extracted from STRING (version 10; www.string-db.org). PPI networks were constructed and visualized using Cytoscape (version 2.8; http://www.cytoscape.org/).

Quantitative data are presented as the mean ± SD of triplicates. Statistical analysis was carried out using GraphPad Prism software (version 6; GraphPad Software, Inc.). Multigroup comparisons were analyzed using one-way ANOVA followed by Tukey's post hoc test. The IC₅₀ values of Tan IIA, Tet, and cisplatin were calculated using nonlinear regression. P<0.05 was considered to indicate a statistically significant difference.

Treatment with cisplatin, Tan IIA, and Tet for 48 h inhibited the viability of HEI-OC1 auditory cells in a dose-dependent manner (Fig. 1A-C), with IC₅₀ values of 42.89 µM, 151.80 and 1.04×10³ mg/l, respectively.

To investigate whether Tan IIA and Tet administration could suppress the inhibitory effect of cisplatin on HEI-OC1 auditory cell viability, cells were treated with 30 µM cisplatin combined with Tan IIA (0.2, 0.5, 1.0 and 1.5 mg/l) or Tet (37.5, 75, 125 and 250 mg/l) for 30 h. To minimize drug-related cytotoxicity, the selected concentrations were <IC₅₀ values. The addition of Tan IIA at concentrations <1.5 mg/l augmented the effect of cisplatin and further inhibited viability significantly compared with cisplatin alone (P=0.0018, Fig. 1D). However, at concentrations of 37.5 and 75 mg/l Tet significantly increased viability, compared with cisplatin alone (P<0.0001, Fig. 1E).

The percentage of apoptotic HEI-OC1 auditory cells was significantly increased following cisplatin treatment, compared with control cells (10.27±0.64% and 3.11±0.17% in the second (upper right) and third (lower right) quadrants, respectively; P=0.0002; Fig. 2). However, co-treatment with cisplatin and Tet reduced the apoptosis rate to 7.18±0.33% (P=0.0054). Collectively, these results indicated that low concentrations of Tet could protect HEI-OC1 cells against cisplatin-induced cytotoxicity.

To examine the molecular mechanisms associated with the otoprotective properties of Tet, RNA sequencing (RNA-seq) analysis was carried out on HEI-OC1 auditory cells treated with 30 µM cisplatin alone or combined with 37.5 mg/l Tet. Illumina sequencing produced a total of 518.83 million raw reads, corresponding to 510.65 million clean reads and 74.31 Gbp (Table SI). The average GC content was 50.26%, while the average frequency of reads >Q30 was 95.91%. Transcriptome assembly generated 1,455,306 transcripts. In comparison with control cells, 638 DEGs were identified following cisplatin treatment, including 361 upregulated genes and 277 downregulated genes (Fig. 3A-C). Moreover, 882 DEGs were observed in cells treated with a combination of cisplatin and Tet, including 415 upregulated and 467 downregulated genes (Fig. 3B and C). Thus, relative to untreated cells, the combination of cisplatin and Tet induced more DEGs than cisplatin alone. A total of 449 DEGs were shared between the two datasets, including 221 upregulated and 228 downregulated DEGs (Fig. 3B and C). In comparison with cisplatin alone, the combination with Tet (37.5 mg/l) only induced 31 DEGs, including 26 upregulated and 5 downregulated DEGs). In total, 1,088 DEGs were identified, including 626 non-overlapping DEGs (Fig. 3A-C).

To examine the molecular mechanisms underlying cisplatin-induced cytotoxicity, the biological processes and pathways associated with the identified DEGs were examined using GO and KEGG analysis. GO functional enrichment analysis indicated that downregulated DEGs, such as histone (Hist)1 and Hist2 gene clusters (Fig. 4), were associated with biological processes related to ‘DNA replication-dependent nucleosome assembly’, ‘nucleosome assembly’, and ‘DNA-templated transcription, initiation’ (Table I). Other downregulated DEGs were also associated with biological processes, including ‘negative regulation of apoptotic process’ (thrombospondin1, THBS1; TWIST2; insulin-1 receptor antagonist gene, IL1RN; IGF1) and regulation of cell migration (IL1RN; SERPINE1; IGF binding protein 5, IGFBP5).

Moreover, the downregulated DEGs identified following cisplatin treatment were also associated with several KEGG pathways. Genes in the Hist1 and Hist2 clusters were associated with ‘Systemic lupus erythematosus’, ‘Alcoholism’ and ‘Viral carcinogenesis’ (Table II). IGF1 and THBS1 were involved in ‘PI3K-Akt signaling pathway’, ‘Rap1 signaling pathway’, and ‘HIF-1 signaling pathway’.

Notably, the upregulated DEGs induced by cisplatin in HEI-OC1 auditory cells were significantly associated with autophagy (autophagy-related gene 12, ATG12; ATG9B; ATG2A), apoptosis-associated processes, response to DNA damage and cell cycle arrest. These genes included phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), Bcl-2 binding component 3 (BBC3), zinc finger matrin-type 3 (ZMAT3), p53-induced death domain protein 1 (PIDD1), B-cell translocation gene protein 2 (BTG2), thioredoxin-interacting protein (TNXIP), DNA damage induced apoptosis suppressor (DDIAS), cycle-dependent kinase inhibitor 1A (CDKN1A), transformation related protein 53-induced nuclear protein 1 (TRP53INP1), FOXO3, and Fas (Table I). These DEGs interacted closely (Fig. 4) and some (including CDKN1A, BBC3, ZMAT3, PMAIP1, FAS, PIDD1, and FOXO3) were significantly associated with ‘p53 signaling pathway’ and ‘FoxO signaling pathway’ (Table II).

As indicated in Fig. 3, 450 DEGs were shared between the two treatments. The shared DEGs included Fas, PMAIP1, ATG12, CDKN1A, and ZMAT3, which were upregulated, and THBS1 and SERPINE1, which were downregulated (Fig. 5A-D). Enrichment analysis showed these shared genes enriched in similar functional categories and KEGG pathways as DEGs induced by cisplatin alone (Tables III and IV). The upregulated genes including THBS1, CDKN1A, Fas, PMAIP1, TXNIP, and ZMAT3 were associated with biological processes, such as ‘programmed cell death’ and ‘cell cycle’ (Table III). Genes including CDKN1A, Fas, PMAIP1, TXNIP, and ZMAT3 and pathways including ‘p53 signaling pathway’ and/or ‘Apoptosis’ (Table IV). The downregulated genes including the histone genes, endothelin 1 (EDN1), suppressor of cytokine signaling 3 (SOCS3), and insulin receptor substrate 1 (IRS1) were associated with biological processes, such as ‘nucleosome assembly’ and ‘regulation of phosphate metabolic process’ (Table III). The downregulated DEGs including THBS1, collagen type III alpha 1 chain (COL3A1), and platelet derived growth factor subunit B (PDGFB) were involved in ‘ECM-receptor interaction’ and ‘Focal adhesion’ (Table IV).

Of the 882 DEGs induced by the cisplatin and Tet combination, the 467 downregulated histone genes were involved in biological processes including ‘nucleosome assembly’ and ‘nucleosome organization’ (Table V), and one KEGG pathway of ‘Systemic lupus erythematosus’ (Table VI). The downregulated DEGs that were specifically induced by combination treatment, including cyclin-dependent kinase inhibitor 2A (CDKN2A) were related to biological processes like ‘rRNA processing’ and ‘ncRNA metabolic process’ (Table V). The downregulated CCND1 specifically responded to combination treatment was also involved in ‘Focal adhesion’ (Table VI).

The 415 upregulated genes (such as ZMAT3, PMAIP1, TRP53INP1, CDKN1A, BTG2, and FAS) were clustered in biological processes including ‘programmed cell death’, ‘cell cycle’, ‘induction of apoptosis’ and ‘apoptosis’ (Table V), as were the DEGs that were specifically upregulated by the combination treatment (including Caspase-3 (CASP3) and IL18 (Table V). Genes like CDKN1A, CASP3, ZMAT3, PMAIP1, and FAS were involved in ‘p53 signaling pathway’, and genes including CASP3, CDKN1A, and FAS were related to ‘pathways in cancer’, as were TCF7L1 and FZD6 genes that were specifically responded to combination treatment (Table VI).

Most of the 432 DEGs that were specifically induced by the combination treatment were associated with the biological processes and KEGG pathways that were similar to the functional categories associated with the 882 DEGs related to combination treatment (Table SII). For instance, downregulated genes including CDKN2A were related to the biological processes that related to the metabolism and biogenesis of ribosome and nucleosome, and the processing of RNAs (Table SII). upregulated CASP3 and IL18, were associated with ‘positive regulation of apoptotic process’ and ‘positive regulation of cell death’. The ‘Pathways in cancer’ pathway enriched upregulated WNT9A, TCF7L1, and FZD6 (Table SII).

The expression patterns of several DEGs are shown in Fig. 5. The FPKM of ATG12, ATG2A, ZMAT3, DDIAS, TRP53INP1, TXNIP, CDKN1A, BTG2, PMAIP1, PIDD1 and Fas increased following treatment with cisplatin alone or in combination with Tet, compared with untreated control cells. By contrast, serpine1, THBS1 and IGFBP5 were downregulated.

The expression of IGF1 (Fig. 5B) decreased following cisplatin treatment but was rescued by the addition of Tet (P<0.05). FOXO3 expression followed the opposite pattern (Fig. 5C). In comparison with control and cisplatin alone, Tet treatment increased the expression of caspase 3, IGF2R, FZD6, IL18, TCF7L1, and decreased the expression of WNT2/4, CCND1 and CDKN2A (Fig. 5B and C). The gene expression changes induced specifically by Tet, and their associated biological processes, may serve an important role in inhibiting cisplatin-induced cytotoxicity in HEI-OC1 auditory cells.

Protein expression of several DEGs was determined using western blot analysis. CDKN1A/p21 and Fas upregulation was cisplatin-dependent and Tet-independent (Fig. 5E). However, expression of CDKN2A/p16-INK4A and WNT2 were decreased, whereas TCF7L1, FZD6, and CASP3 were specifically upregulated by the combination of cisplatin and Tet.