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Identification of microRNA targets in the mammalian inner ear using a comprehensive transcriptome and proteome integrated approach

ABSTRACT: We have employed a novel approach for the identification of functionally important microRNA (miRNA)-target interactions using integrated miRNA, transcriptome and proteome profiles with advanced in silico analysis. By looking at both the transcript and protein levels of expression, a thorough coverage of miRNA regulation was obtained. Microdissected auditory and vestibular sensory epithelia were used as the model system, thus being the first time such a comparison was carried out in a neuroepithelial system. Moreover, this is one of only a few studies employing proteome screening for the identification of miRNA targets. Notably, this approach can be employed for the study of other tissues and organs. We detected the expression of 157 miRNAs in the inner ear sensory epithelia, with 53 miRNAs differentially expressed between the cochlea and vestibule. By searching for enrichment and depletion of miRNA targets in the transcript and protein datasets with a reciprocal or similar expression, respectively, as the regulatory miRNA, we identified functionally important miRNAs. Finally, the interaction between miR-135b and PSIP1-P75, a transcriptional coacitvator previously unknown in the inner ear, was identified and validated experimentally. We suggest that miR-135b may serve as a cellular effector, involved in regulating some of the differences between the cochlear and vestibular hair cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE23081 | GEO | 2011/04/20

SECONDARY ACCESSION(S): PRJNA131695

REPOSITORIES: GEO

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Project description:We have employed a novel approach for the identification of functionally important microRNA (miRNA)-target interactions using integrated miRNA, transcriptome and proteome profiles with advanced in silico analysis. By looking at both the transcript and protein levels of expression, a thorough coverage of miRNA regulation was obtained. Microdissected auditory and vestibular sensory epithelia were used as the model system, thus being the first time such a comparison was carried out in a neuroepithelial system. Moreover, this is one of only a few studies employing proteome screening for the identification of miRNA targets. Notably, this approach can be employed for the study of other tissues and organs. We detected the expression of 157 miRNAs in the inner ear sensory epithelia, with 53 miRNAs differentially expressed between the cochlea and vestibule. By searching for enrichment and depletion of miRNA targets in the transcript and protein datasets with a reciprocal or similar expression, respectively, as the regulatory miRNA, we identified functionally important miRNAs. Finally, the interaction between miR-135b and PSIP1-P75, a transcriptional coacitvator previously unknown in the inner ear, was identified and validated experimentally. We suggest that miR-135b may serve as a cellular effector, involved in regulating some of the differences between the cochlear and vestibular hair cells. We investigated the mRNA expression profile of the cochlear and vestibular sensory epithelia from inner ears of postnatal day 2 mice using the Affymetrix GeneChip® 430 2Mouse Genome array. Cochlear and vestibular sensory epithelia were dissected from wild type C3H mice and collected separately. The vestibular epithelia consisted of the saccule, utricle and the lateral and anterior cristae. Both the cochlear and vestibular sensory epithelia were dissected with their underlying mesenchyma. Altogether three pools, three biological replicates, of each tissue type were collected consisting of cochlear or vestibular sensory epithelia dissected from 10 to 12 inner ears.

Project description:MicroRNAs (miRNAs) inhibit the translation of target mRNAs and affect, directly or indirectly, the expression of a large portion of the protein-coding genes. This study focuses on miRNAs that are expressed in the mouse cochlea and vestibule, the two inner ear compartments. To identify miRNAs that are expressed in the vertebrate developing inner ear, we used miRNA microarrays. Similar miRNA profiles were found in newborn (P0) mouse whole cochleae and vestibules. 105 miRNAs were found to be expressed in the whole P0 cochlea and 114 miRNAs expressed in the whole P0 vestibule with average intensities higher than twice the global background, out of 206 included in the arrays. Only 24 miRNAs were found to have different levels of expression in these whole organs, and these differences were mild (15-40%). The microarray results were intersected with two bioinformatic complementary approaches in order to choose candidate miRNAs that are predicted to be expressed specifically in the inner ear sensory epithelia (see the paper). Six small (<40 nt) RNA samples were hybridized with 4 microarrays (to save costs, some of the microarrays were hybridized with two different samples, labeled with different CyDyes â Cy3 and Cy5). Each RNA sample contains small RNAs (<40 nt) pooled from at least 20 newborn cochleae or vestibules, and there are 3 different samples for each organ. However, one of the samples (WT_P0_whole_Cochlea_3_V4) was hybridized with a different array than the other samples (printed from the same probes, but at a different date), and therefore excluded from the analysis described in the paper. Each microarray contains at least 4 identical spots per each probe that may be considered as technical replicates. Four of the samples were hybridized in parallel at the same day to two microarrays, and the dyes were swaped: First microarray: WT_P0_whole_Cochlea_1 (Cy3) [GSM388075] and WT_P0_whole_Vestibule_2 (Cy5) [GSM388635]; second microarray: WT_P0_whole_Cochlea_2 (Cy5) [GSM388633] and WT_P0_whole_Vestibule_1 (Cy3) [GSM388634]. The other 2 samples: WT_P0_whole_Vestibule_3 (Cy3) [GSM388637] and WT_P0_whole_Cochlea_3_V4 (Cy3) [GSM388660] were hybridized to two individual microarrays, at different dates. The miRNA profile in the cochlear and vestibular samples were compared: the vestibular samples are considered as the âexperimentâ samples, and the cochlear samples â as âreferenceâ. Positive control probes (labeled as âcontrol 1â in raw data) were added to RNA samples before labeling. The average of empty and buffer spots was used to calculate the global background of each array, and only miRNAs with an average expression higher than twice the fold of the array global background at least in one of the tissues were considered as expressed.

Project description:The inner ear in mammals is derived from a simple ectodermal thickening called the otic placode. Through a series of complex morphological changes, the placode forms the mature inner ear comprising of the auditory organ (cochlea) and the vestibular/balance organs (utricle, saccule, and three semi-circular canals). The vast majority of genes known to be involved during inner ear development have been found through mutational screens or by chance. To identify genes that can serve as novel candidates required for inner ear development, and also candidate genes for uncloned human deafnesses, inner ear tissues from mouse embryos from E9 to E15 were microdissected and expression-profiled at half-day intervals. Also profiled was the non-inner ear mesenchymal tissue surrounding the inner ear tissue. Various patterns of gene expression were identified, and significant biological pathways that these genes represented were identified. Also identified were mouse genes whose human orthologs are located within uncloned non-syndromic deafness intervals, thus serving as candidates for sequence analysis. Experiment Overall Design: Inner ear tissues from E9 to E15 were microdissected at half-day intervals. E9 is the earliest stage when the otic placode is clearly visible and able to be microdissected cleanly. E15 is the stage when all the organs of the inner ear have become established, as have the sensory hair and non-sensory support cells within those organs. For each of the stages from E9 to E10, whole inner ears were profiled. For each of the stages from E10.5 to E12, the primordial cochlear and vestibular organs were profiled separately. For each of the stages from E12.5 to E15, the cochlea and the saccule were profiled separately, whereas the utricle and the three ampullae were combined and profiled together. Any given tissue from any given stage was a collection of anywhere between 4 to 17 identical tissues, and was obtained in duplicate (i.e. from different litters). Hence, a total of 58 inner ear samples were obtained. Moreover, non-inner ear tissue found in the immediate vicinity of inner ear tissue was also obtained and profiled. Specifically, all non-inner ear tissue from E9 was profiled in duplicate. Non-inner ear tissue from E9.5 to E10.5 was pooled and profiled together (in duplicate), whereas that from E11 to E15 was pooled and profiled together (also in duplicate). Therefore, a total of 6 non-inner samples were obtained.

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Identification of microRNA targets in the mammalian inner ear using a comprehensive transcriptome and proteome integrated approach

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