G3BP1-associated post-transcriptional regulation of MUC16 under hyperosmotic stress contributes to corneal epithelial...

Dry eye disease (DED) is characterized by tear hyperosmolarity, which disrupts corneal epithelial homeostasis and barrier integrity. Membrane-associated mucin 16 (MUC16) is a major component of the ocular surface glycocalyx; however, the post-transcriptional mechanisms linking hyperosmotic stress to MUC16 suppression remain incompletely understood. This study investigated whether the RNA-binding protein Ras-GTPase-activating protein-binding protein 1 (G3BP1), a central component of stress granules (SGs), contributes to MUC16 dysregulation and corneal epithelial barrier dysfunction under hyperosmotic stress.

Immortalized human corneal epithelial cells (HCECs) were exposed to hyperosmotic media (450-550 mOsm/kg). G3BP1 expression, SG assembly, MUC16 mRNA and protein expression, and MUC16 mRNA stability were evaluated using quantitative real-time PCR, western blotting, immunofluorescence, and actinomycin D chase assays. G3BP1 was silenced using siRNA, and G3BP1-MUC16 RNA association was assessed by RNA immunoprecipitation followed by quantitative PCR (RIP-qPCR). Corneal epithelial barrier function was evaluated by transepithelial electrical resistance, sodium fluorescein permeability, and analysis of ZO-1 and Occludin. Computational docking and molecular dynamics simulations were used to investigate the structural plausibility and dynamic stability of a predicted G3BP1-MUC16 RNA interaction.

Hyperosmotic stress increased G3BP1 mRNA and protein expression and promoted the formation of cytoplasmic G3BP1/TIA-1-positive puncta consistent with SG assembly. In parallel, MUC16 mRNA and protein expression were reduced, showing an inverse association with G3BP1 levels. G3BP1 knockdown partially restored MUC16 expression and transcript stability under hyperosmotic conditions. RIP-qPCR demonstrated enrichment of MUC16 transcripts in G3BP1 immunoprecipitates, supporting an association between G3BP1-containing ribonucleoprotein complexes and MUC16 mRNA. Hyperosmotic stress impaired epithelial barrier function, as indicated by reduced transepithelial electrical resistance, increased fluorescein permeability, and disruption of tight-junction-associated proteins; these alterations were partially improved by G3BP1 silencing. Computational analyses supported the structural plausibility and dynamic stability of the predicted protein-RNA complex.

Hyperosmotic stress is associated with increased G3BP1 expression, stress granule assembly, and post-transcriptional suppression of MUC16 in corneal epithelial cells. The findings support a G3BP1-dependent contribution to MUC16 transcript destabilization and epithelial barrier dysfunction. However, whether stress granule assembly is required for MUC16 repression and whether G3BP1 directly binds MUC16 RNA through the predicted structural interface require further experimental validation.