A Cell Type-Specific Approach to Elucidate the Role of miR-96 in Inner Ear Hair Cells
Mutations in miRNA-96, a microRNA expressed within the hair cells (HCs) of the inner ear, result in progressive hearing loss in both mouse models and humans. While previous studies have delved into miR-96 transcriptional cascades via whole organ of Corti microarray experiments of diminuendo (Mir96Dmdo) mice, they face limitations in pinpointing cell type-specific differentially expressed genes. This hinders the ability to conclusively determine if the effects of Mir96Dmdo are specifically within HCs and contribute to the observed abnormal Mir96Dmdo HC phenotype and determine the role of miR-96 in HCs. In this study, we generate the first HC-specific RNA-sequencing (RNA-seq) datasets from Mir96Dmdo; Atoh1/nGFP+ postnatal day 1 wildtype, heterozygous, and homozygous mutant mice. Our differential gene expression analysis between Mir96Dmdo homozygous mutant HCs compared to wildtype HCs identified 215 upregulated and 428 downregulated genes. Many significantly downregulated genes in Mir96Dmdo homozygous mutant HCs have established roles in HC development and/or known roles in deafness such as Myo15, Myo7a, Ush1c Gfi1, and Ptprq, some of which were not previously identified in other miR-96 datasets. In addition, active modules of   protein-protein interaction networks of significantly downregulated genes in Mir96Dmdo homozygous mutant HCs reveal enrichment in GO terms with biological functions such as sound perception and endocytosis. Genes significantly upregulated in Mir96Dmdo homozygous mutant HCs, which are more likely to be direct targets of miR-96, show higher expression in wildtype supporting cells compared to wildtype HCs, suggesting a role of miR-96 in suppressing non-HC genes during HC development. Finally, all generated HC-specific Mir96Dmdo RNA-seq datasets from this manuscript are now publicly available in the miR-96 specific gEAR profile (https://umgear.org/p?l=miR96). Overall design: To investigate the role of miR-96 and further understand the transcriptional cascade of miR-96 in inner ear hair cells, we performed bulk RNA-sequencing on pooled cochlear and vestibular hair cells of  postnatal day (P) 1 wildtype, Mir96Dmdo heterozygous, and Mir96Dmdo homozgyous mutant mice (n=3 mice per genotype). Differential gene expression analysis was performed on Mir96Dmdo homozgyous mutant hair cells compared to wildtype controls.