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. 2014 Jun 18;15(1):484.
doi: 10.1186/1471-2164-15-484.

Next-generation Sequencing of Small RNAs From Inner Ear Sensory Epithelium Identifies microRNAs and Defines Regulatory Pathways

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Free PMC article

Next-generation Sequencing of Small RNAs From Inner Ear Sensory Epithelium Identifies microRNAs and Defines Regulatory Pathways

Anya Rudnicki et al. BMC Genomics. .
Free PMC article

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figures

Figure 1
Figure 1
miRNA expression similarity in the cochlear and vestibular sensory epithelia. A. Tissue similarity and common expression of inner ear miRNAs. High similarity of miRNA expression in the cochlear and vestibular sensory epithelium can be observed with the majority (455) of the 529 miRNAs, expressed with an RPM > 1, in both tissues. B. The relative proportion of each miRNA among the most highly expressed miRNAs in each tissue. The total amount (100%) represents the sum of RPK values of each miRNA. The Y-axis represents the proportion of each miRNA out of this total. Each miRNA is color-coded and indicated in the legend.
Figure 2
Figure 2
The genomic location, predicted structure and relative level of expression of miR-6715-3p and miR-6715-5p. A. pre-miR6715 is located in the second intron of the Tectb gene. B. miRDeep2 prediction of the RNA secondary hairpin structure of the unprocessed miR-6715-3p, miR-6715-5p, and loop. The seed region of miR-6715-3p is indicated in blue and of miR-6715-5p in green. C. Middle density plot shows the distribution of reads in the predicted precursor sequence, as produced by miRDeep2.
Figure 3
Figure 3
miR-6715-3p and miR-6715-5p expression in the inner ear. A. qRT-PCR analysis and validation of novel miRNAs in the mouse cochlea sensory epithelium. miR-6715-3p and miR-6715-5p exhibited increased expression with age between E16 and P8 (*) P < 0.05, (**) P < 0.005. n = 5. B. In situ hybridization of P0 mouse cochlear and vestibular sections. miR-6715-3p and miR-6715-5p expression was found in the hair and supporting cells of both cochlea and vestibule, spiral and vestibular ganglia, stria vascularis, basilar membrane and in Reissner's membrane. sa, saccule; ut, utricle; sg, spiral ganglia; sm, scala media; st, scala tympani; sv, scala vestibule; ohc, outer hair cells; ihc, inner hair cells; hc, hair cells. Bar: 100 μm.
Figure 4
Figure 4
miRNA-target prediction validation and expression. Using TargetScan, potential targets of miR- 6715-3p and miR-6715-5p were identified, based on the complimentarily of the seed region and 6-7 nt sequence of the target mRNA. To test the possibility of miR-target regulation, luciferase assays were performed. A. Protocadherin19 as a potential target of miR-6715-5p. Wild-type 3’UTR had approximately 65% of luciferase activity as compared to the mutant. (*) P < 0.05. B. Vezatin as a potential target of miR-6715-3p. Wild-type 3’UTR had approximately 45% of luciferase activity as compared to the mutant (*) P < 0.05. C. Activator for the Rho-type GTPases Arhgap12 as a potential target of miR- 6715-3p. Wild-type 3’UTR had approximately 85% of luciferase activity as compared to the mutant. (*) P < 0.05. D. Scn8a is not a direct target for miR-6715-3p, as the luciferase activity was not reduced.
Figure 5
Figure 5
qRT-PCR analysis of Arhgap12 expression in the mouse cochlea sensory epithelia. Arhgap12 exhibited a similar expression pattern at E16, P0 and at P8 in cochlear sensory epithelia, with no significant differences between ages. n = 5.
Figure 6
Figure 6
Arhgap12 protein expression in mouse inner ear sections. Immunohistochemistry of Arhgap12 (green) in P0 inner ear sections, with hair cells stained by myosin VI (red) and nuclei stained by Draq5 (blue). A. Arhgap12 was detected in the organ of Corti, in hair and supporting cells, stria vascularis, spiral ligament and Reissner’s membrane. B. Magnification of the organ of Corti, with localization of Arhgap12 in epithelial cells. C. Arhgap12 is localized to the spiral ganglia. D. In the vestibular system, Arhgap12 is present in hair and supporting cells in the crista. Bar: A, 100 μm; B-D, 50 μm.
Figure 7
Figure 7
Arhgap12 protein expression in mouse inner ear whole-mount preparations. Immunohistochemistry of Arhgap12 (green) in whole mount preparations of P0 mouse cochleas, with cell junctions stained by ZO-1 (red) and actin stained by phalloidin (blue). A. Arhgap12 is localized to the hair and supporting cells of the sensory epithelium. B. In a higher magnification of A, Arhgap12 is localized to the hair bundle and the kinocilium. C. Additional plane showing mutual exclusion of localization of Arhagp12 and ZO-1. hc, hair cells. Bar: A, 10 μm; B, 5 μm.
Figure 8
Figure 8
A newly identified miRNA-target regulatory pathway in the inner ear. A suggested pathway for miR-6715-3p and Arhgap12, whereby miR-6715-3p down-regulates expression of Arhgap12, which in turn blocks the hydrolysis of Rac1 (Ras-Related C3 Botulinum Toxin Substrate 1) GTPase. This may result in increased cell adhesion, actin reorganization and inner ear morphogenesis.

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