Abstract
Hearing loss impacts a significant portion of the human population. According to a 2018 report by the World Health Organization, about 466 million people worldwide, including 34 million children, experience disabling hearing loss. Recent advances in molecular biology have emphasized the pivotal role of microRNAs in regulating gene expression during the development and maintenance of the inner ear. These small non-coding RNAs influence key biological processes such as cell differentiation, apoptosis, and oxidative stress, which are essential for maintaining cochlear function and protecting auditory sensory cells. Changes in microRNA expression have been linked to different types of hearing loss, including age-related, noise-induced, and genetic or congenital. Our lab focuses on the miRNA-183 family, which includes three microRNAs: miR-183, miR-96, and miR-182. These microRNAs are expressed in sensory neurons and sensory epithelial hair cells. The miRNA-183 family has been linked to hereditary deafness in both humans and mice.To investigate the potential roles of the miR-183 family in the inner ear, our research team conducted bulk RNA sequencing to compare newborn mouse inner ear tissues from wild-type versus miR-183 family knockout (KO) mice. Bulk RNA sequencing revealed numerous gene expression changes in the cochlear sensory epithelium and spiral ganglion due to miR-183 family loss-of-function. However, these changes in gene expression represent an average across the entire tissue and do not provide insight into cell-specific gene expression. To address this, I combined existing bulk RNA sequencing data with publicly available single-cell RNA sequencing data from the gene Expression Analysis Resource (gEAR) database to obtain cellular resolution. First, we compiled a list of differentially expressed predicted miRNA target genes, either upregulated or downregulated, that changed in expression by at least 20% due to miR-183 family KO. From this list, we selected the top 25 highly expressed genes and the top 25 most changed genes, whether upregulated or downregulated. Subsequently, we generated heatmaps in gEAR to visualize and explore cell-specific gene expression patterns of these genes across different cochlear cell types and spiral ganglion neurons.
Results revealed 31 Genes of Interest (GOIs) based on hair cell specificity in the sensory epithelia at P1 and 37 GOIs based on developmental expression in the spiral ganglion from E14 to P1, representing a total of 60 individual genes. Concurrently, the human gene database GeneCards was used to gather information on the names, functions, and associated diseases, including hearing loss, of these 60 genes. Furthermore, DAVID (Database for Annotation, Visualization, and Integrated Discovery) was also utilized to perform Gene Ontology (GO) analysis, which enhanced the classification of these genes and helped identify patterns related to their potential functions in the mouse inner ear. After this thorough analysis, a total of 19 GOIs were identified: Egr1, Nnat, Plod2, Rims3, Cntn1, Idh2, Cfl1, Anp32b, Palld, Hspa2, Kif5a, Map1b, Zbtb20, Plag1, Prkce, Amotl2, Slc44a2, Dcx, and Sncb. These 19 genes stood out due to their expression patterns after KO, their cell-specific expression in single-cell analysis data, their potential involvement in hearing loss-related diseases, and their specific cellular processes (i.e., GO terms). Out of these 19 GOIs, four genes including Egr1, Nnat, Plod2, and Rims3 were selected as easily validatable targets to investigate cell-specific changes in expression resulting from miR-183 family KO by immunostaining. Collectively, these findings could advance hearing research by illustrating the impact of miR-183 family KO on specific genes involved in the development and maintenance of the inner ear and their potential effects on hearing loss.