USH2A knockdown induces apoptosis and reduces prestin expression in HEI-OC1 hair cell model

Mutations in the USH2A gene are strongly associated with hereditary sensorineural loss. However, the cellular mechanisms linking USH2A deficiency to cochlear hair cell vulnerability remain incompletely understood. This study aimed to investigate the effects of USH2A knockdown on Prestin expression and apoptosis-related signaling in a cochlear hair cell model. Stable USH2A knockdown HEI-OC1 cells were generated using lentiviral shRNA transduction followed by puromycin selection (2 μg/mL for 7 days). Gene and protein expression levels of Prestin, Bax, and Bcl-2 were assessed by RT-qPCR (2^(-ΔΔCt) method), Western blotting, and immunofluorescence. Apoptosis was evaluated by flow cytometry (Annexin V/PI), TUNEL staining, and cleaved caspase-3 immunofluorescence. USH2A knockdown reduced Prestin mRNA and protein expression by approximately 60% compared with the sh-NC group (P < 0.01). Bax expression increased more than 2.5-fold, whereas Bcl-2 levels decreased by ∼50%, indicating disruption of apoptotic balance. Flow cytometry revealed increased early apoptotic (2.16% → 4.58%) and late apoptotic (16.3% → 22.0%) populations. TUNEL-positive cells increased from 2.8 ± 0.6% to 12.4 ± 2.1% (P < 0.01), and cleaved caspase-3 fluorescence intensity was approximately threefold higher in the knockdown group. USH2A knockdown significantly reduces Prestin expression and promotes apoptosis through modulation of the Bax/Bcl-2 axis in HEI-OC1 cells. These findings provide mechanistic insights into how USH2A deficiency may increase cochlear hair cell vulnerability and potentially contribute to auditory dysfunction. Further in vivo validation is warranted.