Changes in the dorsal cochlear nucleus (DCN) following exposure to noise play an important role in the development of tinnitus. As the development of several diseases is known to be associated with microRNAs (miRNAs/miRs), the aim of the present study was to identify the miRNAs that may be implicated in pathogenic changes in the DCN, resulting in tinnitus. A previously developed tinnitus animal model was used for this study. The study consisted of four stages, including identification of candidate miRNAs involved in tinnitus development using miRNA microarray analysis, validation of miRNA expression using reverse transcription-quantitative PCR (RT-qPCR), evaluation of the effects of candidate miRNA overexpression on tinnitus development through injection of a candidate miRNA mimic or mimic negative control, and target prediction of candidate miRNAs using mRNA microarray analysis and western blotting. The miRNA microarray and RT-qPCR analyses revealed that miR-375-3p expression was significantly reduced in the tinnitus group compared with that in the non-tinnitus group. Additionally, miR-375-3p overexpression via injection of miR-375-3p mimic reduced the proportion of animals with persistent tinnitus. Based on mRNA microarray and western blot analyses, connective tissue growth factor (
Tinnitus, a condition that affects 7-25% of the population worldwide, is a phenomenon in which sound is perceived in the absence of sound stimuli (
A lack of clarity on the mechanisms underlying the development of tinnitus makes it difficult to design an effective treatment strategy. Therefore, the elucidation of the underlying mechanisms will contribute significantly towards identifying a cure for tinnitus. Maladaptive auditory-somatosensory plasticity in the dorsal cochlear nucleus (DCN) after hearing loss has been suggested as one of the mechanisms promoting the development of tinnitus (
MicroRNAs (miRNAs/miRs) are small non-coding RNAs that regulate gene expression via translational inhibition or mRNA degradation (
Accordingly, the present study was undertaken to identify miRNAs that may be implicated in the pathogenesis of tinnitus. miRNA levels were compared in animal models with and without tinnitus following induction of TTS using microarray analysis and reverse transcription-quantitative PCR (RT-qPCR). Additionally, the candidate miRNAs were overexpressed to examine the differences in the expression of their candidate targets. Specifically, the expression levels of miR-375-3p in the DCNs of animals with tinnitus were measured, and the role of miR-375-3p in tinnitus and the involvement of connective tissue growth factor (
The present study was approved by the Institutional Animal Care and Use Committee of Chung-Ang University (2016-00092). All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (
A total of 102 rats were used in the present study. The study was conducted in four stages (
A total of 8 rats were used in the first stage of the experiment. At 3 weeks post-noise exposure, a total of 5 rats exhibited evidence of tinnitus. Subsequently, 3 rats were randomly selected each from the tinnitus (n=5) and non-tinnitus (n=3) groups. The right DCNs were harvested from these rats, following the protocol outlined in the atlas of Paxinos and Watson (
ABR recordings were performed as described previously (
GPIAS recordings were performed as described previously (
Acoustic stimulation was carried out using sound waves of 2 kHz bandwidth and 60 dB SPL; the center frequencies of 7, 11 and 15 kHz were used as the background noise. A broadband noise burst of 105 dB SPL for a duration of 50 msec served as the startle stimulus. During each session, 15 gap-conditioned stimuli and 15 non-gap-conditioned stimuli were presented in a random pair order. The gap pre-pulse that occurred in each gap-conditioned stimulus was presented 100 msec before the onset of the startle stimulus and lasted for 50 msec. The time interval between the presentation of acoustic stimulations was altered randomly between 17 and 23 sec. The gap-conditioned response/non-gap-conditioned response (G/N) ratios were calculated according to the following equation:
Microarray analysis of the miRNAs was performed at BioCore Co., Ltd. using the Affymetrix miRNA 4.0 microarray (Affymetrix; Thermo Fisher Scientific, Inc.), which contained all the miRNAs in the miRBase Release 20 database (
Total RNA was extracted from the DCN samples using the QIAzol Lysis Reagent (Qiagen GmbH) according to the manufacturer's instructions. The RNA concentration of the samples was determined using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA synthesis was performed using the miScript II RT kit (Qiagen GmbH), and qPCR was performed with primers for miR-15b-3p (cat. no. YP00205898), -105 (cat. no. YP00205105), -221-3p (cat. no. YP00204532), -375-3p (cat. no. YP00204362), -455-5p (cat. no. YP00204363), -544-5p (cat. no. YP02116293), -708-5p (cat. no. YP00204490) and -759 (cat. no. YP00206000; all from Qiagen GmbH) in a Bio-Rad CFX 96 real-time system (Bio-Rad Laboratories, Inc.) using the miScript SYBR Green PCR kit (Qiagen GmbH) as follows: Initial heat activation at 95°C for 2 min, followed by 40 cycles of 95°C for 10 sec and 56°C for 1 min. All PCR reactions were performed under standard PCR conditions; U6 (cat. no. YP00203907; Qiagen GmbH) was used as the endogenous control. The relative quantification (RQ) values were calculated from the quantification cycle (Cq) values using the 2−ΔΔCq method (
Based on the results of miRNA microarray and RT-qPCR analyses, miR-375-3p was selected as the candidate miRNA. To evaluate its role, miR-375-3p mimic (5′-UUUGUUCGUUCGGCUCGCGUGA-3′) (Qiagen GmbH) and miR-375-3p mimic negative control (Qiagen GmbH) were administered to 31 and 28 rats, respectively, that exhibited evidence of tinnitus at 1 week post-noise exposure. Once the rats were anesthetized using the method previously described, and placed in a stereotaxic frame, 5
To identify the target mRNA regulated by miR-375-3p, mRNA microarray analysis was performed on the DCN samples from 3 rats randomly selected each from the mimic non-tinnitus and control tinnitus groups. Microarray analysis of the mRNAs was performed at BioCore Co., Ltd. Briefly, RNA was isolated and prepared as detailed above. The cDNAs were generated using the GeneChip Whole Transcript PLUS Reagent kit (Affymetrix; Thermo Fisher Scientific, Inc.) and labeled with terminal deoxynucleotidyl transferase (TDT) using the Affymetrix proprietary DNA Labeling Reagent (Affymetrix; Thermo Fisher Scientific, Inc.). The labeled samples were hybridized to the GeneChip RaGene 2.0 ST Array (Affymetrix; Thermo Fisher Scientific, Inc.). All arrays were scanned using the Affymetrix GeneChip Scanner 3000, and raw analysis was performed using the Transcriptome Analysis Console™ software. The CEL files generated were imported into the Gene Expression Workflow in GeneSpring GX version 14.9.1 (Agilent Technologies, Inc.). Subsequently, the microarray data were analyzed as described above.
Western blotting was performed to detect the expression levels of the candidate target genes obtained using mRNA microarray and miRNA target prediction server (TargetScan) analyses. In addition, western blotting was performed to determine the expression levels of the candidate target genes along with
Western blotting was carried out on the DCNs, as described previously (
Statistical analysis was performed using the IBM SPSS 21.0 software (IBM Corp.). The ABR thresholds were assessed using the two-way repeated measures ANOVA followed by Tukey's post hoc test. The RQ values obtained from the RT-qPCR and western blot analyses results were examined using the Mann-Whitney U-test. The effect of miR-375-3p overexpression was analyzed using the Pearson's χ2 test. P<0.05 indicated statistically significant differences.
Prior to noise exposure, the ABR thresholds on both the right and left sides of all rats ranged between 20 and 30 dB SPL, at all frequencies, with no significant difference detected between the tinnitus and non-tinnitus groups at all stages. In all groups and at all frequencies, the ABR thresholds on the right side on day 1 after noise exposure were significantly higher compared with those at all other time points. After 1 week of noise exposure, the ABR threshold was significantly lower compared with that on day 1, but significantly higher compared with the baseline threshold and the threshold 3 weeks later. However, there was no significant difference between the baseline ABR threshold and the ABR threshold after 3 weeks of exposure to noise. The ABR thresholds on the left side ranged between 20 and 30 dB SPL. At all time points post-exposure, the measured ABR thresholds at each frequency did not differ significantly between the two groups at the first and second stages during the experiments (
Among individual rats, the pre-exposure G/N ratios ranged from 30 to 70%. Compared with the no-gap condition, all rats showed significant decreases in startle responses under the gap condition at all frequencies (P<0.05). As stated previously, the GPIAS responses were recorded at 3 weeks following noise exposure. In the first stage of the experiment, 5 of the 8 rats examined exhibited no significant decrease in the startle response under the gap condition at one or more frequencies (P>0.05). Consequently, these rats manifested behavioral evidence of tinnitus. The remaining rats exhibited significant decreases in startle responses under the gap condition at all frequencies; hence, they manifested no behavioral evidence of tinnitus. In the second stage of the experiment, 7 of the 13 rats examined exhibited no significant decrease in startle response under the gap condition at one or more frequencies (P>0.05). Thus, these rats manifested behavioral evidence of tinnitus. The remaining rats exhibited significant decreases in startle responses under the gap condition at all frequencies, and were accordingly considered to manifest no behavioral evidence of tinnitus.
Candidate miRNAs were selected based on the results obtained from microarray analysis (non-tinnitus group, n=3; tinnitus group, n=3). First, miRNAs not expressed in humans were excluded. Subsequently, the remaining miRNAs that satisfied the following criterion were selected: Log-ratio intensity >0.379 or <−0.379 (P<0.08) between the tinnitus and non-tinnitus groups, as determined by the Student's t-test. Using this criterion, miR-15b-3p, -105, -221-3p, -375-3p, -455-5p, -544-5p, -708-5p and -759 were selected as candidate miRNAs (
To validate the candidate miRNAs, RT-qPCR was performed (non-tinnitus group, n=6; tinnitus group, n=7) to identify those miRNAs showing a significant difference in RQ values in the tinnitus and non-tinnitus groups. It was observed that, among all the candidate miRNAs, the RQ value of miR-375-3p was significantly decreased in the tinnitus group compared with that in the non-tinnitus group (P=0.028), while the RQ values of miR-15b-3p, -105, -455-5p, -544-5p and -708-5p were not significantly different (
To evaluate the role of miR-375-3p in the development of tinnitus, a miR-375-3p mimic or a mimic negative control was injected into the lateral ventricles of rats with tinnitus at 1 week post-noise exposure. Two weeks later (i.e., 3 weeks post-noise exposure), GPIAS recordings were performed to determine whether tinnitus persisted. It was found that tinnitus persisted in 21 of the 28 rats (75.0%) injected with the miR-375-3p mimic negative control, and in 14 of the 31 rats (45.2%) injected with the miR-375-3p mimic. A total of 17 rats (54.8%) exhibited no tinnitus at 3 weeks post-noise exposure (
To discern the probable targets regulated by miR-375-3p, mRNA microarray analysis was performed (
Along with these genes,
Although numerous studies have investigated the probable causal factors contributing to the development of tinnitus (
It is well known that the majority of genes are regulated by miRNAs, which are non-coding RNAs that modify gene expression via post-transcriptional regulation. Moreover, one single miRNA can regulate the expression of multiple genes, thereby serving as a checkpoint for disease outbreaks. miRNAs are particularly abundant in the brain and play important roles in the development and functioning of neuronal networks, including the regulation of neurogenesis, synaptogenesis and morphogenesis (
In our previous study, a tinnitus animal model was developed to elucidate the mechanisms underlying tinnitus resulting from noise exposure and inducing TTS in rats (
Several researchers have reported the effects of miR-375 on the brain, although it is found in multiple organs or tissues (
Our previous study demonstrated that, subsequent to hearing loss, the auditory projections degrade quickly and more severely in the tinnitus group compared with the non-tinnitus group, thereby leading to a significant increase in the somatosensory projections. This has been identified as an important process in the development of tinnitus (
There were certain limitations to the present study that must be acknowledged. Only male rats were selected in this study. The rats were ensured to be an identical strain and within a certain age range to reduce variabilities. Furthermore, ABR recordings were checked before noise exposure to prove that the hearing did not differ between the groups. Interestingly, female rats do not display greater variability during the reproductive cycle compared with male rats; furthermore, male and female mice and humans also have similar levels of variability in terms of gene expression (
To the best of our knowledge, no previous studies have examined the involvement of miRNAs in the development of tinnitus. In the present study, the expression of miR-375-3p was found to be reduced in the DCNs of rats with tinnitus, and the overexpression of miR-375-3p prevented the persistence of tinnitus by reducing the expression of
The raw data of microRNA microarray analysis generated during the current study are available in the Gene Expression Omnibus (GEO) (accession numbers: GSE172259), [
MC designed the experiments and study. KHH, HC, KRH and MC performed the experiments. MC, KHH and SKM have seen and confirm the authenticity of the raw data. MC and KHH wrote the manuscript, analyzed and interpreted the data. SKM, YKK and IP contributed to designing the experiment and revising the manuscript. All the authors have read and approved the final manuscript.
All procedures for animal care and use were approved by the Institutional Animal Care and Use Committee of Chung-Ang University (2016-00092).
Not applicable.
The authors declare that they have no competing interests.
Not applicable.
Experimental design. TTS, temporary threshold shift; GPIAS, gap pre-pulse inhibition of acoustic startle reflex; ABR, auditory brainstem response; RT-qPCR, reverse transcription-quantitative PCR.
ABR thresholds before and after noise exposure in the first and second stages. Stage one: (A) Non-tinnitus group (n=3) and (B) tinnitus group (n=5). Stage two: (C) Non-tinnitus group (n=6) and (D) tinnitus group (n=7). There were no significant differences in the ABR thresholds of the rats in the tinnitus and non-tinnitus groups at all time points tested. Data are presented as mean ± standard deviation, as determined using two-way repeated measures ANOVA. *P<0.05 compared with baseline; †P<0.05 compared with week 1; and ‡P<0.05 compared with week 3. ABR, auditory brainstem response; SPL, sound pressure level.
ABR thresholds before and after noise exposure in the third stage. (A) Non-tinnitus (n=7) and (B) tinnitus (n=21) groups with miR-375-3p mimic negative control injection. (C) Non-tinnitus (n=17) and (D) tinnitus (n=14) groups with miR-375-3p mimic injection. There were no significant differences in the ABR thresholds among the rats in all groups at all time points tested. Data are presented as mean ± standard deviation, as determined using two-way repeated measures ANOVA. *P<0.05 compared with baseline, †P<0.05 compared with week 1 and ‡P<0.05 compared with week 3. ABR, auditory brainstem response; SPL, sound pressure level.
Reverse transcription-quantitative PCR of candidate miRNAs. The RQ value of miR-375-3p was significantly decreased in the tinnitus group (n=7) compared with that in the non-tinnitus group (n=6). The experiment was repeated thrice. *P<0.05 vs. non-tinnitus group, as determined using the Mann-Whitney test. The bars indicate standard error. RQ, relative quantification; miRNA/miR, microRNA.
Ratio of rats with or without tinnitus post-injection. miR-375-3p mimic and miR-375-3p mimic negative control were administered to 31 and 28 rats, respectively, that exhibited evidence of tinnitus at 1 week post-noise exposure. Two weeks later (i.e., 3 weeks after noise exposure), the GPIAS responses were measured to determine whether tinnitus persisted. The mimic-injected group exhibited a significantly lower ratio of animals with persistent tinnitus at 3 weeks post-noise exposure, as determined using the Pearson's χ2 test (P=0.020, odds ratio =0.275, 95% confidence interval: 0.090-0.833). The number of animals in each experimental group are labeled in the graph. GPIAS, gap pre-pulse inhibition of acoustic startle reflex; miR, microRNA.
Representative western blots and quantitative analyses of CTGF, INHBA, HOXA2, and KCNAB3 expression levels. CTGF level was significantly lower in the mimic non-tinnitus group compared with that in the control tinnitus group (P=0.028). Data are presented as mean ± standard error, as determined using the Mann-Whitney test. *P<0.05. CTGF, connective tissue growth factor; INHBA, inhibin β-A; HOXA2, homeobox A2; KCNAB3, potassium voltage-gated channel subfamily A regulatory beta subunit 3.
Candidate microRNAs selected based on microarray analysis.
Candidate microRNAs | Log-ratio | P-value |
---|---|---|
Log-ratio between the tinnitus and non-tinnitus groups: | ||
>0.379 (P<0.08) | ||
miR-15b-3p | 0.456 | 0.020 |
miR-221-3p | 0.500 | 0.033 |
miR-455-5p | 0.394 | 0.076 |
miR-544-5p | 0.666 | 0.012 |
miR-708-5p | 0.773 | 0.043 |
Log-ratio between the tinnitus and non-tinnitus groups: | ||
<−0.379 (P<0.08) | ||
miR-105 | −0.519 | 0.030 |
miR-375-3p | −0.530 | 0.059 |
miR-759 | −0.661 | 0.058 |
miR, microRNA. |
Changes in GPIAS responses in the mimic non-tinnitus group wherein tinnitus ceased following miR-375-3p mimic injection.
No. | Background noise (kHz)
| |||||
---|---|---|---|---|---|---|
6-8
|
10-12
|
14-16
| ||||
G/N ratio |
P-value | G/N ratio |
P-value | G/N ratio |
P-value | |
1 | ||||||
Baseline | 0.567 | 0.002 | 0.331 | <0.001 | 0.284 | <0.001 |
Week 1 | 1.100 | 0.793 | 0.360 | 0.001 | 0.559 | 0.223 |
Week 3 | 0.599 | <0.001 | 0.373 | 0.001 | 0.665 | 0.024 |
2 | ||||||
Baseline | 0.477 | 0.001 | 0.571 | 0.028 | 0.249 | <0.001 |
Week 1 | 0.980 | 0.497 | 0.782 | 0.093 | 0.323 | 0.002 |
Week 3 | 0.176 | <0.001 | 0.222 | 0.010 | 0.397 | 0.014 |
3 | ||||||
Baseline | 0.728 | <0.001 | 0.771 | 0.006 | 0.701 | <0.001 |
Week 1 | 0.730 | <0.001 | 0.972 | 0.908 | 0.960 | 0.315 |
Week 3 | 0.785 | 0.006 | 0.785 | 0.003 | 0.842 | 0.015 |
4 | ||||||
Baseline | 0.638 | <0.001 | 0.567 | 0.001 | 0.568 | 0.005 |
Week 1 | 0.984 | 0.627 | 0.744 | 0.010 | 0.972 | 0.576 |
Week 3 | 0.692 | 0.005 | 0.714 | 0.044 | 0.608 | <0.001 |
5 | ||||||
Baseline | 0.537 | 0.001 | 0.295 | 0.001 | 0.296 | <0.001 |
Week 1 | 0.246 | 0.002 | 0.966 | 0.106 | 0.289 | 0.001 |
Week 3 | 0.247 | 0.002 | 0.329 | 0.003 | 0.964 | 0.020 |
6 | ||||||
Baseline | 0.583 | 0.002 | 0.532 | <0.001 | 0.679 | 0.011 |
Week 1 | 0.970 | 0.852 | 1.031 | 0.890 | 0.623 | 0.003 |
Week 3 | 0.734 | 0.033 | 0.692 | 0.005 | 0.602 | 0.004 |
7 | ||||||
Baseline | 0.749 | 0.045 | 0.368 | <0.001 | 0.451 | 0.001 |
Week 1 | 0.401 | <0.001 | 0.703 | 0.014 | 1.015 | 0.633 |
Week 3 | 0.734 | 0.015 | 0.642 | 0.026 | 0.421 | 0.001 |
8 | ||||||
Baseline | 0.121 | <0.001 | 0.357 | <0.001 | 0.443 | 0.015 |
Week 1 | 1.092 | 0.663 | 0.258 | <0.001 | 0.148 | <0.001 |
Week 3 | 0.154 | <0.001 | 0.108 | <0.001 | 0.101 | <0.001 |
9 | ||||||
Baseline | 0.332 | <0.001 | 0.424 | <0.001 | 0.266 | <0.001 |
Week 1 | 0.497 | <0.001 | 0.583 | 0.013 | 0.667 | 0.254 |
Week 3 | 0.480 | <0.001 | 0.354 | <0.001 | 0.284 | 0.013 |
10 | ||||||
Baseline | 0.285 | 0.001 | 0.373 | 0.002 | 0.558 | 0.001 |
Week 1 | 0.378 | <0.001 | 0.765 | 0.178 | 0.990 | 0.760 |
Week 3 | 0.343 | <0.001 | 0.343 | 0.016 | 0.533 | 0.010 |
11 | ||||||
Baseline | 0.624 | 0.006 | 0.642 | 0.029 | 0.526 | 0.001 |
Week 1 | 1.026 | 0.020 | 1.006 | 0.443 | 0.669 | 0.036 |
Week 3 | 0.378 | 0.002 | 0.564 | 0.011 | 0.327 | 0.008 |
12 | ||||||
Baseline | 0.579 | 0.006 | 0.300 | <0.001 | 0.199 | <0.001 |
Week 1 | 1.077 | 0.358 | 0.858 | 0.310 | 0.519 | 0.002 |
Week 3 | 0.616 | 0.001 | 0.555 | 0.007 | 0.448 | 0.040 |
13 | ||||||
Baseline | 0.595 | 0.001 | 0.452 | 0.002 | 0.358 | 0.001 |
Week 1 | 0.589 | 0.001 | 0.561 | 0.012 | 0.812 | 0.101 |
Week 3 | 0.479 | <0.001 | 0.723 | 0.036 | 0.514 | 0.001 |
14 | ||||||
Baseline | 0.453 | <0.001 | 0.669 | 0.038 | 0.668 | 0.012 |
Week 1 | 0.440 | <0.001 | 0.623 | 0.004 | 0.770 | 0.165 |
Week 3 | 0.421 | 0.007 | 0.345 | <0.001 | 0.498 | 0.001 |
15 | ||||||
Baseline | 0.455 | 0.001 | 0.507 | 0.024 | 0.379 | 0.001 |
Week 1 | 1.311 | 0.102 | 0.705 | 0.254 | 0.310 | 0.004 |
Week 3 | 0.518 | 0.024 | 0.502 | 0.021 | 0.433 | 0.025 |
16 | ||||||
Baseline | 0.560 | 0.017 | 0.620 | 0.014 | 0.312 | <0.001 |
Week 1 | 0.286 | 0.047 | 0.596 | 0.141 | 0.532 | 0.006 |
Week 3 | 0.511 | <0.001 | 0.362 | <0.001 | 0.483 | 0.002 |
17 | ||||||
Baseline | 0.594 | 0.005 | 0.486 | <0.001 | 0.611 | 0.021 |
Week 1 | 1.061 | 0.818 | 0.727 | 0.044 | 0.618 | 0.036 |
Week 3 | 0.467 | <0.001 | 0.650 | 0.040 | 0.741 | 0.049 |
RMS-GSR/RMS-NGSR. RMS, root-mean-square; GSR, gap-conditioned startle response; NGSR, non-gap-conditioned startle response; GPIAS, gap pre-pulse inhibition of acoustic startle reflex.
Candidate target genes of microRNA-375-3p selected based on microarray analysis.
Candidate target genes | Log-ratio between the mimic non-tinnitus and control tinnitus groups: <−0.585 (P<0.25) | P-value |
---|---|---|
KCNAB3 | −0.650 | 0.136 |
INHBA | −0.709 | 0.166 |
HOXA2 | −0.957 | 0.240 |