Abstract
Damage to mechanosensory hair cells (HCs) of the inner ear leads to permanent hearing loss. Small RNAs, namely endogenous small interfering RNAs (endo-siRNAs) and canonical microRNAs, are known to affect HC development and maintenance. microRNA biogenesis requires both Dgcr8 and Dicer1, whereas siRNA biogenesis requires only Dicer1. Conditional knockout (CKO) of Dgcr8 shows HC aberrations and mild HC loss at 2 weeks of age, whereas Dicer1 CKO exhibits less HC aberrations or loss at 2 weeks of age. Thus we hypothesize there is a greater depletion of miRNAs in the inner ear of mice with HC-specific Dgcr8 CKO compared to mice with HC-specific Dicer1 CKO. |HC-specific Dgcr8 CKO and Dicer1 CKO mice were generated using Atoh1-Cre. Total RNA was isolated from the inner ears of 2 biological replicates from Dgcr8 CKO, Dicer1 CKO, and control mice. Small RNA content was examined by Illumina small RNA sequencing. Small RNA content was compared between CKO and control inner ear samples. Further examination of normalized read values included determination of microRNAs and potential endo-siRNAs that exhibited at least 2-fold differences in abundance between CKO and control inner ears, a comparison of microRNA content to previously published studies, miRNA cluster analysis, an evaluation of microRNA/host gene co-transcription, validation of a subset of miRNAs by qRT-PCR, and visualization of change between control and CKO cochlea for a selected few microRNAs by in situ hybridization (ISH). |For Dgcr8 CKO inner ear versus control, there was 1 downregulated and no upregulated microRNAs with a ≥ 2-fold statistically significant change in expression. In contrast Dicer1 CKO inner ear showed 25 downregulated and 11 upregulated microRNAs. Notably, microRNA-96, a known HC-specific microRNA, was significantly downregulated in both CKO groups. Potential endo-siRNAs showed relatively low abundance compared to microRNAs and were unchanged in Dgcr8 CKO inner ear, whereas there were 19 downregulated and 20 upregulated potential endo-siRNAs in Dicer1 CKO inner ear. Further assessments of small RNA sequencing data largely validated the subset of microRNAs that were highly abundant within the inner ear. The fold change identified by qRT-PCR, for a subset of miRNAs, was not statistically significant. ISH revealed a depletion in HC and SE-specific miRNA in both CKO mice compared to control. |Our analyses suggest that Dgcr8 may not only affect miRNAs but may also affect other classes of RNAs, such as mRNAs, compared to Dicer1. Ambiguous quantitative and qualitative results do now allow for defining the contribution of miRNAs on the more affected HC phenotype observed in Dgcr8 CKO mice compared to Dicer1 CKO mice. Investigation of both CKO mice utilizing cell specific dissection followed by small RNA sequencing may provide further insight towards the contribution of miRNAs to the HC phenotype observed.