The Role of MicroRNA181a in Avian Auditory Hair Cell Regeneration

Auditory hair cells, the sensory transducing cells of the inner ear, regenerate after injury in birds but not mammals. Microarrays were used to examine gene expression differences between proliferating and quiescent chicken auditory epithelia, as well as expression gradients along the tonotopic axis of the uninjured inner ear. Differential expression of a number of genes was confirmed by quantitative PCR (qPCR). Gene set enrichment analyses of the microarray data identified myriad microRNAs (miRNA) that were predicted to be activated in the proliferating inner ear, some of which are also predicted to be differentially active along the tonotopic axis of the basilar papilla (BP), the avian counterpart of the organ of Corti. One miRNA in particular, miR181a, was selected for functional validation and was found to stimulate proliferation with production of some new hair cells when overexpressed in chicken cochlear duct expiants. Further, BPs treated with forskolin, which stimulates proliferation without injury, and transfected with anti-miR181a to knock down endogenous miR181a have fewer dividing cells as assayed by BrdU labeling than BPs exposed to forskolin and transfected with a nontargeting miRNA. This observation suggests miR181a mediates forskolin induced proliferation. MiR181a was also found to play a key role in hair cell regeneration after injury, as BPs cultured with streptomycin had less BrdU labeling when they were also transfected with anti-miR181a. Importantly, BPs transfected with anti-miR181a and exposed to streptomycin did not have a reduction in the number of cells labeling for the early cell death marker activated caspase-3 when they were also transfected with anti-miR181a, suggesting that anti-miR181a transfection actually interferes with the regenerative process rather than preventing cell death. Surprisingly, miR181a expression did not increase with streptomycin exposure when assessed by qPCR, suggesting that this miRNA's role in regeneration is mediated by increased activity rather than expression. Of the miRNA computationally predicted to possibly play a role in regeneration, miR17, miR19a, miR30a, and miR183 were also selected for functional experiments based on what is known about their function in other systems. Of these, miR19a and miR183 were also found to stimulate proliferation when expressed in the inner ear. Clearly, miRNA in general, and miR181a in particular, play an important role in hair cell regeneration in the avian inner ear.