Identification of constitutively expressed miRNAs in specific tissues has been a major focus of miRNA investigations 
. While numerous studies have profiled miRNA expression in mouse inner ears, miRNA expression in the rat cochlea has not been examined. Here, we successfully detected a group of miRNAs in both normal and noise-exposed sensory epithelia in the rat cochlea.
The current study reveals constitutive expression of miRNAs in the normal cochlear sensory epithelium and noise-induced changes in the expression of those miRNAs during the acute phase of cochlear pathogenesis. The changes in expression are time-dependent. Bioinformatic analysis identifies the potential target genes with apoptotic properties for the seven significantly-downregulated miRNAs that have been well characterized. Further expression analysis of the predicted target genes reveals an inverse relationship between the expression levels of miR-183 and Taok1. This relationship was further confirmed by manipulation of miR-183 expression in cochlear organotypic cultures. Together, these observations implicate miRNAs as potential players in the regulation of cochlear responses to acoustic trauma.
Previously, Weston et al. (2006) screened the expression of 344 mature miRNAs in the whole inner ear of the developing mouse (P0–P100). Of the 344 mature miRNAs, 102 miRNAs were expressed from P0–P36. Many of the identified miRNAs were subsequently confirmed in a recent investigation by Wang et al. (2010), who showed the expression of 122 miRNAs at E13.5 and 199 miRNAs at E16.5. More recently, Elkan-Miller et al. (2011) examined the expression of 586 miRNAs and detected 138 miRNAs in the P2 mouse cochlea. Here, we examined the expression of 378 miRNAs and detected 176 miRNAs in the adult rat cochlear sensory epithelium. Among the 178 miRNAs, miRs 135a, 136, 139-5p, 146a, 146b, 155, 184, 186, 187, 188-5p, 190b, 193b, 203, 210, 224, 291a-3p, 296-5p, 301a, 301b, 323-3p, 324-3p, 329, 34b-3p, 350, 369-3p, 375, 376a, 376c, 384-5p, 411, 433, 434-5p, 434-3p, 532-3p, 598, 667, 668, 672, 682, 708, 744, 758, and 872 have not been previously identified in a mouse model 
. These miRNAs may be species specific for the rat cochlea. However, their detection may also be due to our enriched sensory epithelium samples compared to the whole cochlear samples used in previous studies, or due to the higher sensitivity of the qRT-PCR technique used in our miRNA assessment compared with the microarray analysis used in previous investigations 
We identified miRNAs 182, 183 and 96 in the rat, which are three intensively investigated miRNAs that are present in mouse ears 
. Thus, this cluster of miRNAs seems to be conserved between species. We found a significant downregulation of miR-183 at 1 d post-noise exposure. However, the expression levels of the other 2 miRNAs were not significantly altered. This differential expression pattern has been observed in previous studies and has been attributed, in part, to variation in the rate of miRNA degradation 
. Thus, there is a possibility that the lack of change in miRs-182 and -96 following acoustic trauma is due to a slower degradation rate or no degradation compared to the targeted degradation of miR-183, which in turn may lead to the inconsistent expression pattern of these miRNAs within the cluster.
In the present investigation, we found a time-dependent alteration in miRNA expression post-noise exposure. At 2 h post-noise exposure, only one miRNA exhibited a significant change in expression. In contrast, there was an increase in the number of miRNAs that were altered at 1 d post-noise exposure. This temporal pattern of changes in expression is likely to be related to the progression of sensory cell degeneration post-noise exposure. As we reported earlier, sensory cell lesions grow in a time-dependent manner 
. The growth of the lesion is expected to provoke more cells to undergo the degenerative process and consequently, more miRNAs to undergo changes in expression.
Another possible contributor to the temporal change in miRNA expression is differences in damaging initiators during the different phases of cochlear pathogenesis. Acute damage to cochlear tissues observed 2 h post-noise exposure is primarily associated with the mechanical stress caused by physical disturbances to the cochlear structure during the period of noise exposure. In contrast, subsequent pathologies, including energy exhaustion, 
oxidative stress, 
and ionic imbalance, 
may be the result of a metabolic disruption. These metabolic disruptions are likely to cause changes in miRNA expression through different mechanisms than those caused by acute mechanical stress.
In our study, the majority of miRNAs detected at 1 d post-noise exposure were significantly downregulated when compared to their constitutive expression levels. This finding of a downregulation dominated change is consistent with previous observations of oxidative stress-related changes in miRNA expression in cultured cells derived from the organ of Corti 
as well as in non-cochlear apoptotic models 
. As miRNAs act as inhibitors of mRNA in controlling cellular processes 
a reduction in miRNA expression following acoustic trauma may lead to an increase in the expression of mRNA targets.
Many of the target genes of miRNAs that underwent changes in expression, as revealed by our bioinformatic analysis, have been linked to sensorineural hearing loss. For example, Xiap
is a predicted target of miR-186. Previous studies have shown the involvement of this target gene in protection against noise-induced hearing loss when it is over-expressed in transgenic mice 
, a predicted target of miR-124, has been suggested to be involved in stress-related pathways in the auditory system 
and also to be linked to cochlear apoptosis induced by acoustic trauma 
, a predicted target of miR-124, was recently identified to be upregulated 2 h post-noise exposure in the chinchilla cochlea and was linked to the p38/MAPK signaling pathway 
, a predicted anti-apoptotic target of miRs 124 and 381, is required for cell survival, and inhibition of Bcl11b
both in vitro
and in vivo
leads to apoptosis 
. Recently, Bcl11b
was associated with age-related hearing loss and was suggested to be required for OHC survival and normal hearing 
. Thus, miRNA/mRNA target pairs may be present in the cochlea and may be involved in regulating apoptosis-related pathways. To further our understanding of noise-induced apoptosis in the cochlea, it will be important to identify these miRNA/mRNA target pairs. Furthermore, it will be important to understand the biological significance of these target pairs and their relationship with sensory cell damage following noise exposure.
One of the possible miRNA/mRNA target pairs revealed by the current study is Taok1/
was identified by our bioinformatic analysis and its association with miR-183 was experimentally verified. Taok1
contains two binding sites for miR-183 in its 3′untranslated region. Both sites are perfectly complementary to the miR-183 sequence in the miRNA seed region. In non-cochlear tissues, Taok1
has been associated with activation of the mitogen-activated protein kinase pathway in response to stress and DNA damage 
. In cancerous tissues, Taok1
has been found to activate the c-Jun N-terminal kinase mitogen-activated protein kinase pathway 
. In human neuroblastoma cells, Taok1 transfection induces apoptosis 
. In noise-damaged cochleae, the mitogen-activated protein kinase pathway has been linked to cochlear apoptosis and inhibition of this pathway reduces apoptosis 
. These observations suggest the involvement of Taok1
in the regulation of cochlear responses to acoustic trauma, possibly through the regulation of apoptosis. Bioinformatic analysis also revealed other targets of miR-183, including Egr1
. Previously, acoustic overstimulation (125 dB SPL) in the rat cochlea was found to increase the transcriptional expression of Egr1
, which further led to an increase in its protein expression 
. Irs1 modulates insulin signaling pathways and has not been previously identified in the cochlea. However, a previous study has shown Irs2-deficient mice to exhibit sensorineural hearing loss 
. Thus, it would be important to study whether Irs1 also plays a role in regulating cochlear responses to acoustic trauma.
The current study documents the constitutive expression pattern of 176 miRNAs in the normal rat cochlear sensory epithelium and noise-induced changes in the expression of these miRNAs. The changes in expression are time-specific. Further target prediction analysis and subsequent experimental verification revealed the miR-183/Taok1 target pair. These results implicate miRNAs as regulators of noise-induced cochlear responses to acoustic trauma. The discovery of differentially expressed miRNAs after noise exposure in the rat cochlea can assist with the future exploration of miRNA/mRNA target pairs that may be manipulated to reduce noise-induced cochlear damage.