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

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.本研究采用一种全新方法，通过整合微小RNA（microRNA，miRNA）、转录组与蛋白质组谱图，并结合先进的计算机硅基分析（in silico analysis），鉴定具有功能重要性的miRNA靶标相互作用。通过同步检测转录本与蛋白质的表达水平，本研究实现了对miRNA调控的全面覆盖分析。本研究以显微切割获取的听觉及前庭感觉上皮作为模型系统，这也是首次在神经上皮系统中开展此类比较研究。此外，本研究属于少数采用蛋白质组筛选策略鉴定miRNA靶标的研究之一。值得注意的是，该方法可推广应用于其他组织与器官的相关研究。本研究在内耳感觉上皮中检测到157种miRNA的表达，其中53种miRNA在耳蜗与前庭组织间存在差异表达。通过在转录组与蛋白质组数据集中分别筛选与调控miRNA呈反向表达或同向表达的miRNA靶标的富集与缺失特征，本研究鉴定出具有功能重要性的miRNA。最终，本研究鉴定并通过实验验证了miR-135b与PSIP1-P75的相互作用，后者是一种此前在内耳中未被报道的转录共激活因子。本研究推测miR-135b可作为细胞效应因子，参与调控耳蜗毛细胞与前庭毛细胞之间的部分功能差异。本研究采用Affymetrix GeneChip® 430 2小鼠全基因组芯片，检测了出生后第2天小鼠内耳耳蜗及前庭感觉上皮的mRNA表达谱。研究对象为野生型C3H小鼠，研究人员分别切割获取其耳蜗与前庭感觉上皮并单独收集。前庭上皮包含球囊、椭圆囊以及外侧与前半规管壶腹嵴。耳蜗与前庭感觉上皮均连同其下方的间充质一同被切割获取。每种组织类型均设置3个生物学重复样本池，每个样本池由10~12个内耳切割获取的耳蜗或前庭感觉上皮混合制备而成。