Next-generation sequencing of small RNAs from inner ear sensory epithelium identifies microRNAs and defines regulatory pathways
- Anya Rudnicki†1,
- Ofer Isakov†2,
- Kathy Ushakov1,
- Shaked Shivatzki1,
- Inbal Weiss1, 3,
- Lilach M Friedman1,
- Noam Shomron2 and
- Karen B Avraham1Email author
© Rudnicki et al.; licensee BioMed Central Ltd. 2014
Received: 9 March 2014
Accepted: 13 June 2014
Published: 18 June 2014
The mammalian inner ear contains sensory organs, the organ of Corti in the cochlea and cristae and maculae in the vestibule, with each comprised of patterned sensory epithelia that are responsible for hearing and balance. The development, cell fate, patterning, and innervation of both the sensory and nonsensory regions of the inner ear are governed by tight regulation involving, among others, transcription factors and microRNAs (miRNAs). In humans, mutations in specific miRNA genes are associated with hearing loss. In mice, experimental reduction or mutations of miRNAs in the inner ear leads to severe developmental and structural abnormalities. A comprehensive identification of miRNAs in the sensory epithelia and their gene targets will enable pathways of auditory and vestibular function to be defined.
In this study, we used Next-Generation Sequencing (NGS) to identify the most prominent miRNAs in the inner ear and to define miRNA-target pairs that form pathways crucial for the function of the sensory epithelial cells. NGS of RNA from inner ear sensory epithelial cells led to the identification of 455 miRNAs in both cochlear and vestibular sensory epithelium, with 30 and 44 miRNAs found in only cochlea or vestibule, respectively. miR-6715-3p and miR-6715-5p were defined for the first time in the inner ear. Gene targets were identified for each of these miRNAs, including Arhgap12, a GTPase activating protein, for miR-6715-3p, implicating this miRNA in sensory hair cell bundle development, actin reorganization, cell adhesion and inner ear morphogenesis.
This study provides a comprehensive atlas of miRNAs in the inner ear sensory epithelia. The results provide further support of the essential regulatory role of miRNAs in inner ear sensory epithelia and in regulating pathways that define development and growth of these cells.
KeywordsDeafness Inner ear Sensory epithelia RNA-seq MicroRNAs
miRNAs play an essential role in inner ear development . miRNAs are small non-coding RNAs that regulate gene expression post-transcriptionally. By binding to sequences in the 3’ untranslated region (UTR) of mRNAs, a miRNA can inhibit target mRNAs by translational suppression and mRNA destabilization . miRNAs have been implicated in hearing loss in humans, since mutations in the seed region of miR-96 or its gene are associated with hearing loss in three extended families [3, 4]. miRNAs were also reported in other human ear pathologies, including an elevation of miR-21 in cholesteatomas  and in vestibular schwannomas . miRNAs were found to regulate the otitis media inflammatory response . Their study in humans, however, has been hampered by the unavailability of inner ear RNA from human subjects, making the mouse an invaluable model for studying miRNA development and regulation in the inner ear . In an ENU-induced mutant mouse exhibiting deafness and vestibular dysfunction, a mutation in the miR-96 seed region was found to be responsible for hearing loss, presumably due to the alteration in expression and hence function of both direct and indirect gene targets . A number of mouse mutants affecting miRNA regulation through Dicer have been created, leading to developmental inner ear defects [10, 11]. These conditional knock-out mouse mutants have been instrumental in demonstrating that miRNAs are vital for inner ear morphogenesis and development of the sensory epithelia and sensory neurons.
Expression of over 300 miRNAs have been reported in mouse and rat inner ears thus far, identified by microarray analysis [8, 10, 12, 13], with the majority of work performed on the triad mir-96, −182, and −183 . In order to further identify and characterize miRNAs in the mammalian inner ear, we used NGS for the first time to identify miRNAs in cochlear and vestibular sensory epithelia. Using this method, we identified over 500 inner ear miRNAs, both in common between and unique to each tissue. We further validated and characterized the expression of two miRNAs, miR-6715-3p and miR-6715-5p, in the mouse inner ear. Vezatin, an adherens junctions transmembrane protein previously implicated in deafness  and Arhgap12, a Rho GTPase activating protein , were verified to be gene targets of miR-6715-3p. Protocadherin 19, a member of the δ2 subclass of nonclustered protocadherins and associated with epilepsy and mental retardation , is a gene target of miR-6715-5p. Arhgap12, newly described in the inner ear, is expressed in the epithelial cell-cell junctions of the hair and supporting cells. The miR-6715-3p–Arhgap12 miRNA target pair defines a new regulatory pathway. Understanding these interactions may shed light on known and novel miRNAs, their effect on the development of normal and impaired hearing, and the mechanisms leading towards deafness.
RNA-seq derived miRNA transcription profile of the mouse inner ear sensory epithelium
Most highly expressed miRNAs in cochlear and vestibular sensory epithelium
Read count in cochlear sample
Read count in vestibular sample
Differentially expressed miRNAs in cochlear and vestibular sensory epithelium
Read count cochlea
Read count vestibule
Fold change (1 > 2)
Fold change (2 > 1)
Statistic (Chi^2 dist)
Corrected P-value (Bonferroni)
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
0.00E + 00
Targets for most highly expressed miRNAs
Predicted mRNA targets for the most abundant miRNAs in the sensory epithelia of the inner ear
Bdnf, Ednrb, Rdx, Rere, Sox2
Chd7, Grid1, Psap, Slc19a2, Tnfrsf11b
Atf2, Fbxo11, Gabrb3, Kcnq4, Rb1, Rere, Slc12a2, Slc19a2
Identification of inner ear miRNAs
Expression of new inner ear miRNAs
In order to explore the spatial expression pattern of the two inner ear miRNAs, in situ hybridization analysis was performed on inner ears derived from P0 C57/BL6 mice, at the same age the RNA-seq was performed. Consistent with the qRT-PCR results, expression of the two miRNAs was observed in P0 mice (Figure 3B). A clear staining of both miRNAs was observed in many parts of the inner ear, in spiral and vestibular ganglia, basilar membrane and Reissner's membrane, the stria vascularis and spiral ligament. In a higher magnification, in the cochlea, expression of both miRNAs was observed in the organ of Corti, in the spiral limbus and in particular in the hair and supporting cells. In the vestibular system, staining was observed in sensory epithelia of the saccule and the utricle, in hair and supporting cells.
Target pathways in auditory and vestibular function
The function of miRNAs and their targets in the mammalian inner ear are now being discovered, as the link between miRNAs and hearing and deafness has been established. The inner ear is a complex tissue with multiple cell types, with miRNAs involved in fine-tuning the multitude pathways required for functional hearing. While there are a relatively small number of miRNAs in mammals, their role in regulation far surpasses their number, since each miRNA can exert its effect on hundreds of downstream targets . Hundreds of miRNAs have been discovered in the inner ear; only a subset of these miRNAs have validated targets (reviewed in Table 1).
Our study was designed to profile the general expression of miRNAs in the inner ear and a comparison of the auditory and vestibular epithelia. We chose the time point of p0 since work by our group [10, 13] and others have demonstrated that miRNAs are expressed and functional at this stage, including miRNA-96, which is essential for hearing [3, 9]. RNA-seq analysis led to the identification of over 500 miRNAs, with miR-182 as the most highly expressed miRNA in both sensory epithelia (Table 1) and accounting for more than 50% of the 20 most highly expressed miRNAs. A few targets have been confirmed for miR-182, including Sox2, Clic5 and Tbx1 [28–30]. The second most abundant miRNA in the cochlear and vestibular sensory epithelia, miR-181a-5p, has no known validated targets in the inner ear. This miRNA has been studied in other systems and found to have a pro-proliferative role in cultured human myeloid leukemia cells  and regulates thymic selection in mouse T-cells . The role of this miRNA has been studied in the avian inner ear, where it has been shown to promote proliferation .
We validated a number of targets for miR-6715-3p and miR-6715-5p. Predicted targets for the miRNAs described in this study have direct or potential implications for hearing loss. Pcdh19 is expressed in the zebrafish inner ear . Mutations in protocadherin 19 (PCDH19) have been associated with X chromosome-linked epilepsy and mental retardation and it is a member of the protocadherin family of proteins . This family includes protocadherin 15 (Pcdh15), with a known function in the tip links of the stereocilia of the inner ear in conjunction with cadherin 23  and has been associated with multiple mutations in both non-syndromic and syndromic hearing loss in humans [35, 36].
Vezatin (Vezt) is an adherens junctions transmembrane protein, and while it is ubiquitously expressed in these junctions, it is especially prevalent in in hair and supporting cells of the mouse inner ear . Vezt mutant mice suffer from late onset progressive hearing loss, due to hair cell apoptosis. Most compelling, the resistance of the organ of Corti to mechanical stress was reduced when vezatin was absent in the inner ears of conditional mutants. These studies led to the prediction that vezation is required to maintain junction integrity and is crucial to protect the junctions from mechanical stress due to sound trauma.
Arhgap12, a GAP protein, is involved in controlling the time period in which Rac1 will remain active. In MDCK cells, Arhgap12 expression overlaps with Rac1 expression [38, 39], probably due to the regulation of Arhgap12 on Rac1 at this time point. In the inner ear, besides the actin-rich areas, Rac1 is localized to hair cells and in particular, the kinocilium . In accordance with our data, Arhgap12, at P0, with expression in the hair cells and kinocilium, might be regulating by Rac1 at this time, when the hair bundle is still developing and needs to be under close regulation. The kinocilium is necessary for proper development  and may have a structural role in guiding the architecture of the hair bundle and/or be involved in G-protein signaling in the kinocilium. If the latter is true, then Arhgap12 may regulate its GTPase activity. In our model of the miR-6715-3p circuit in the inner ear, we propose that the miRNA indirectly enhances the action of Rac1 and therefore may promote hair bundle development, actin reorganization, cell adhesion and inner ear morphogenesis.
The identification of inner ear-related miRNAs by RNA-seq analysis demonstrates that the dataset is reliable not only for characterizing expression profiles of known miRNAs, but also for discovery of novel miRNAs in the inner ear. Further investigation of these miRNAs may shed light on their regulatory roles in various molecular pathways underlying the development of the embryonic inner ear.
All procedures involving animals were approved and met the guidelines described in the National Institutes of Health Guide for the Use of Laboratory Animals and approved by the Animal Care and Use Committee of Tel Aviv University (M-10-087). Cochlear and vestibular sensory epithelia were dissected from 12 inner ears of 6 P0 C57Bl/6 J mice. The cochlear sensory epithelia included the organ of Corti with some attached membranes, while the vestibular sensory epithelia included the saccule, utricle, lateral crista and anterior crista. The dissected sensory epithelia were placed immediately in Qiazol lysis buffer (Qiagen) on ice and stored at −80°C for several hours. The sensory epithelia was then thawed and total RNA was isolated, using the miRNeasy Mini Kit (Qiagen) without DNAse, which enables purification of total RNA of >18 nt. The isolated RNAs from the samples were precipitated in sodium acetate and glycogen in 80% ethanol (Ambion protocol), in order to further clean and concentrate the RNA samples. The precipitated RNA samples were dissolved in nuclease-free water and their quality was assessed by 1.2% agarose gel (90 V).
Twelve cochlear epithelia were pooled and twelve vestibular epithelia were pooled, and each pool was used to create a library for MPS and sequenced. No biological repeats were performed. miRNA libraries for MPS were created using the TruSeq SmallRNA SamplePrep Kit (Illumina), with one modification. Instead of running the samples on acrylamide gel in order to isolate the miRNA band, the samples were loaded on a 4% agarose E-Gel and the purification was performed with the Qiagen Kit. The libraries were multiplexed and 2.5 pM of each library was sequenced at SR × 36 bp using the Illumina Genome Analyzer IIx in the same lane at the Functional Genomics Laboratory at Tel Aviv University.
miRNA sequence analysis
A total of 8,763,589 and 10,048,818 raw sequences of small RNAs were found in the cochlear and vestibular samples, respectively. Small RNA adapter sequences were clipped using fastq-mcf (ea-utils: http://code.google.com/p/ea-utils/), discarding sequences shorter than 16 nucleotides and adapter dimers. Following adapter clipping, 8,340,741 and 9,441,679 reads remained. Differential expression of the resulting sequences was performed using miRNAkey. Specifically, sequence alignment was performed using Burrows-Wheeler Aligner (BWA)  against mouse mature miRNA sequences (downloaded from miRBase (Release 20: June 2013; http://www.mirbase.org/), while allowing one mismatch between read and reference. The read count for each read was normalized using the RPM method. Differential expression was tested using chi-squared proportion testing and corrected using the Bonferroni correction for multiple comparisons. Novel miRNA prediction was performed using miRDeep2 .
Cochlear sensory epithelia of E16, P0 and P8 C57BL/6 mice was dissected. Small RNAs were extracted using the miRNeasy Mini Kit (QIAGEN). Custom-made probes, designed by Applied Biosystems, were used to detect miRNAs. miRNAs and U6B RNA (endogenous control) were reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qRT-PCR reaction was conducted using the FastStart Universal Probe Master (Roche) in the StepOne Plus qRT-PCR machine (Applied Biosystems). All miRNAs expression was normalized to the expression of U6B. At each age, sensory epithelia from 4–5 mice were pooled and five RT-PCR experiments were performed.
In situ hybridization
Inner ears of P0 C57BL/6 mice were dissected and fixed with 4% paraformaldehyde. Whole mount in situ hybridization analysis was performed using the Exiqon protocol. miRCURY LNA™ microRNA detection custom-made probes, labeled with digoxygenin (DIG) (Exiqon), were used for the detection of novel miRNAs. Probes were hybridized with the tissue, 20-22°C below Tm of the probe. Probes were detected with the anti-DIG-AP (alkaline phosphatase conjugated) antibody (Roche), and the color reaction was developed using the NTB/BCIP (Sigma). The ears were frozen and cryosectioned to 10-18 μm sections and mounted. Images were taken using the Zeiss Aviovert200 M microscope. Three experiments were performed, and 3–5 ears were included in each experiment.
miRNA target prediction
Target prediction for all miRNAs was predicted by TargetScan Custom (Release 5.2; http://www.targetscan.org/vert_50/seedmatch.html), TargetScanMouse (Release 5.2, http://www.targetscan.org/mmu_50/) and/or miRanda (http://www.microrna.org/microrna/home.do). Chosen potential targets were compared to gene expression databases (SHIELD; https://shield.hms.harvard.edu/) and known hearing loss genes and loci (Hereditary Hearing Loss Homepage, http://hereditaryhearingloss.org).
3’ UTRs of chosen potential targets were cloned into the pGL3 luciferase reporter vector, downstream to the luciferase reporter gene, creating the 3’UTR-wt-pGL3 vector. The first three nucleotides of the miRNA binding site in the 3’UTR were mutated by site directed mutagenesis, creating the 3’UTR-mut-pGL3 vector. The miR-6715 miRNA expression vector was created by cloning the genomic polycistronic miR-6715-3p and miR-6715-5p into the miRvec expression vector (obtained as a gift from Reuven Agami) . HEK293T cells were transiently transfected with either 3’UTR-wt-pGL3 or with 3’UTR-mut-pGL3, for each of the tested targets, together with the corresponding miRNA expression vector and Renilla expressing vector. The luciferase reporter assay was performed 48 hours following transfection using the Dual-Luciferase® Reporter assay system (Promega). Three experiments were conducted, with duplicates.
Whole mount and paraffin sections of P0 C67/BL6 mouse inner ear were prepared for staining as previously described . Myosin VI was used to stain hair cells, phalloidin to stain actin and Draq5 to stain nuclei. The following antibodies were used: goat-anti-Arhgap12 antibody (Santa Cruz), 1:100; mouse-anti-ZO-1 (Zymed), 1:100; rabbit-anti-myosin VI (Proteus BioSciences) 1:200; and Draq5 1:600 (Abcam). Antibody specificity of Arhgap12 was assessed by a competition assay in HCT116 cells, with blocking by an Arhagap12 peptide (Santa Cruz) (Additional file 6).
For qRT-PCR and luciferase assays, the Student’s two-tailed t test P values of less than 0.05 were considered to be statistically significant and those of less than 0.005 were considered to be highly statistically significant. The data in the figures are presented by mean + SEM. Differential expression of miRNAs between paired samples was measured using a chi-squared statistic. P-values are calculated for the null hypothesis of no differential expression between the two samples. Final P-values were corrected using the Bonferroni correction for multiple hypotheses testing .
Availability of supporting data
The RNA-seq data from this study is available in the NCBI Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra), under accession number SRP043019.
This study was supported by the Israel Science Foundation (grant no. 1320/11), National Institutes of Health (NIDCD) R01DC011835, I-CORE Gene Regulation in Complex Human Disease Center No. 41/11 and a fellowship from the Edmond J. Safra Center for Bioinformatics at Tel Aviv University. We thank Varda Oron-Karni for her aid with RNA-seq.
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