We have employed a novel and generalizable method to efficiently identify functional miRNA-target interactions in a neuroepithelial tissue. This approach can be extended to any tissue or organ of interest. Specifically, we investigated the differences between sensory epithelia of the cochlear and vestibular portions of the inner ear, in an attempt to decipher critical elements driving differential gene regulation in each system.
In this study we describe a miRNA profile of the inner ear sensory epithelia. To date, two studies profiled the miRNAs in the whole inner ear and whole cochlea and vestibule 
. By focusing on a more specific tissue, our work aims to provide depth and understanding of the inner ear organs with a primary sensory role. We report the presence of 157 miRNAs in at least one of the sensory epithelia in the inner ear, with 52 differentially expressed between the cochlea and vestibule. A close analysis of the miRNA differential profile revealed that many of the miRNAs co-expressed in the studied tissues are clustered in the mouse genome. This observation is consistent with the notion that clustered miRNAs are usually expressed together as polycistronic, co-regulated units 
. The vestibular up-regulated miRNAs include all the members of the miR-183/96/182 cluster previously demonstrated to be specifically expressed in the mammalian inner ear hair cells and ganglia 
. As the amount of RNA obtained from the dissected tissues is greater in the cochlea, due to its larger size, the differential expression of these miRNAs might be due to the different percentage of hair cell specific RNA in the total RNA from each of the two tissues.
Specific expression profiles of a miRNA in a given tissue may point to the particular role of the miRNA in that tissue. It has been suggested that miRNAs serve as cell effectors among cells of related fates 
. Furthermore, it is believed that miRNAs play an important role in terminal differentiation and maturation of different cell types within a particular cell lineage, as well as regulating cellular processes in differentiated cells during morphogenesis and homeostasis (reviewed in 
). The cochlear and vestibular sensory epithelia share many similarities and differences. Specifically, the sensory cells embedded in these tissues function through the same mechanotransduction mechanism and have a similar but not identical morphology. Both cell types have stereocilia projections arranged in bundles but the shape and arrangement of the bundles is different in the two systems 
. Importantly, cochlear hair cells are unable to regenerate in the mammal, while early vestibular hair cells are able to do so to some extent 
. We speculate that the miRNAs differentially expressed between the cochlea or vestibule may participate in regulating these tissue identities and maintaining their distinct function.
The present study expands on the known inner ear transcript and protein profiles. We characterized the repertoire of differentially expressed transcripts and proteins in the vestibular system as compared to the cochlea. Several of the genes identified in our analyses were previously studied in the inner ear and a few (e.g., crystallin 
and cochlin 
) have been shown to cause deafness. However, many of the genes found to be expressed in the inner ear sensory epithelia, according to the transcript and protein datasets, have not been identified in the inner ear thus far, and their functional role is yet unknown. Notably absent from the proteomic dataset are hair cell-specific proteins. This is likely due to the limitation of the iTRAQ mass-spec method to identify low abundance proteins. The tissues studied contain different cell types, making it difficult to predict the function of genes and proteins within specific cell types. In order to understand their functional relevance, the proteins identified would have to be studied in depth individually.
The correlation between the vestibule to cochlea ratios of the mRNA and the protein levels was relatively low, though significant. This could be due to the limited protein expression data or a relatively high level of post-transcriptional regulation. Similar correlations between mRNA and protein changes were previously observed in analyses of embryonic mouse brain tissues 
, in gastric cancer cells 
and in the yeast Saccharomyces cerevisiae 
Currently, miRNA target identification is based primarily on computational target predication algorithms. The vast number of targets predicted by these algorithms raises the problem of choosing which of these are worthy for experimental validation. For example, searching for the potential targets of the 52 differentially expressed miRNAs using the TargetScan algorithm led to the identification of 11,031 putative conserved targets. Therefore, to narrow down the targets list and to detect miRNA-target pairs with a higher likelihood for successful validation, we utilized a strategy that combines in silico analysis and experimental techniques.
To analyze enrichment or depletion of miRNA targets we applied the FAME algorithm on our datasets of differentially expressed transcripts and proteins. Genes preferentially co-expressed with a miRNA have evolved to avoid targeting by that miRNA 
. Thus, depletion of targets is expected for genes that are expressed in the same tissue as the miRNA 
. We therefore focused on miRNAs and targets with a reciprocal expression and miRNAs and anti-targets (messages selectively avoiding targeting to a miRNA; see 
) with a similar expression pattern. In some cases, miRNAs and their potential targets were observed to have a similar expression pattern, and not a reciprocal one as expected. Such a phenomenon might be explained by the counter regulation of different posttranscriptional control mechanisms or by miRNA induced translation up-regulation as previously observed for the miRNAs miR-369-3 and let-7 in cell cycle arrest 
. We note that some of the miRNA targets predicted by the analysis could only be detected using our proteomics data, while others were only identified using the transcriptomics data. Thus by looking at both levels of expression we were able to identify the most thorough list of miRNA-target pairs. It should be pointed out that our power is limited by the detection constraints of the proteomics screen, and thus we expect this list to be only partial.
The most notable miRNA for which we identified translationally repressed targets was miR-135b, the miRNA with the highest differential expression in our dataset. miR-135b is located within the first intron of the LEM domain containing 1
) gene. Interestingly, our Affymetrix microarray analysis showed a high expression of Lemd1
in the vestibular sensory epithelia. Therefore, it is likely that miR-135b is transcribed as part of Lemd1
, leading to a similar expression pattern. To better understand miR-135b function in the inner ear, we studied its spatial expression. In situ
hybridization demonstrated specific expression of miR-135b in vestibular hair cells. No such expression was observed in the cochlea, consistent with our microarray and qRT-PCR results. The distinct expression pattern of miR-135b most probably points to a specific regulation mechanism that exists in the vestibular hair cells but not in the cochlear hair cells. To date, the only miRNAs identified demonstrating inner ear hair cell specificity are part of the miR-183/96/182 family 
. Unlike miR-135b, these miRNAs are expressed both in the cochlear and vestibular hair cells.
Of the three putative targets of miR-135b, we chose to further validate the interaction with the P75 isoform of PSIP1. PSIP1 is a transcriptional coactivator involved in neuroepithelial differentiation and neurogenesis 
. In particular, it plays a role in gene regulation in the epithelial cells of the lens and is considered to be involved in cell fate determination 
. Such functions correspond well to possible involvement in the differentiation and maintenance of the sensory epithelia in the inner ear. It is therefore not surprising that PSIP1 is expressed in the inner ear sensory epithelia, as demonstrated in this study. The Psip1
gene is alternatively spliced into two different isoforms; P75
, the larger isoform, and P52 
. Of the two, only P75
contains a sequence within its 3′ UTR with the potential of being targeted by miR-135b. Using qRT-PCR and semi quantitative western blot analysis, we were able to demonstrate inhibition of PSIP1-P75 protein expression in the vestibular sensory epithelia suggesting intervention by a translational regulation mechanism. In vitro
analysis further proved an interaction between miR-135b and PSIP1-P75. Interestingly, the efficiency by which miR-135b silences PSIP1-P75, as identified by our in vitro
analysis, is much higher than previously expected for targets with only a single binding site for a miRNA seed 
. Due to the limitation in efficient transfection of inner ear organotypic cultures, we could not show the direct interaction in vivo
. Taken together, our results demonstrate the regulation of PSIP1-P75 by miR-135b in vestibular hair cells. According to our results, miR-135a also has a higher level of expression in the vestibular as compared to the cochlear sensory epithelia. Due to the similarity between miR-135b and miR-135a, we predict that miR-135a also regulates PSIP1-P75 in the vestibular system.
The overall effect of miR-135b in the inner ear is summarized in . In this scheme, we propose a unique mechanism by which miR-135b down-regulates PSIP1-P75 expression in the vestibular hair cells, whereas it remains relatively high in the cochlea. Thus the effect of PSIP1-P75 transcriptional regulation is more pronounced in the cochlear hair cells, leading to downstream perturbation that possibly influences the cell's identity, differentiation and maintenance. PSIP1-P75 was previously shown to be involved in cell survival 
, protection against stress 
, differentiation 
, cell fate determination 
and is believed to regulate genes involved in development 
. We hypothesize that PSIP1-P75 and miR-135b might play a role in regulating these processes in the cochlear hair cells, whereas in vestibular hair cells they are modulated by other miRNA. By this means, miR-135b might serve as a cellular effector, involved in regulating the differences between the cochlear and vestibular hair cells and thus contributes to their distinct cell identities and maintaining their specific functions. It should be pointed out that these processes involve more intricate mechanisms that have yet to be revealed, including the interplay among different miRNAs and proteins.
Schematic representation of a possible role of miR-135b in the inner ear.
In this work we found evidence for the functional importance of many previously unknown inner ear sensory epithelia miRNAs. We reason that miRNAs differentially expressed between the cochlear and vestibular sensory epithelia may participate in regulating the cellular identities of these tissues and maintaining their distinct morphology and function. Using our target analysis approach, we were able to identify both miRNA targets affected at the mRNA level and ones only affected at the protein level. Significantly, the identification of a bona-fide miRNA-target pair, miR-135b and PSIP1-P75, predicts a role for this pair in inner ear cell survival, protection against stress, differentiation, cell fate determination and development, and may explain differences in regeneration of vestibular vs. cochlear hair cells.