miR-196a protects corneal endothelial cells from senescence by modulating mitochondrial and TGF-β signaling

Maintaining corneal transparency and function relies critically on healthy corneal endothelial cells (CECs), which form a hexagonal monolayer. Dysfunction or loss of these cells, often due to senescence or fibrotic disorders like Fuchs endothelial corneal dystrophy (FECD), can lead to severe visual impairment. Current treatments are often invasive, such as corneal transplantation. Understanding mechanisms that protect CECs from senescence, particularly those involving mitochondrial dysfunction and TGF-β signaling, is crucial for developing less invasive therapies. This study explores the potential of miR-196a as a protective agent against CEC senescence.

Researchers cultured human corneal endothelial cells (CECs) and manipulated miR-196a levels using miR-196a mimics (for overexpression) or inhibitors (for suppression). Senescence was experimentally induced in these cells using transforming growth factor β (TGF-β). Various cellular functions were then assessed: cell viability and proliferation via CCK-8 assay and flow cytometry, gene expression through real-time polymerase chain reaction and Western blotting, and mitochondrial function using JC-1 staining and mitophagy markers. Senescence was specifically detected by senescence-associated β-galactosidase staining, while cytoskeletal changes and signaling pathway activation were visualized and quantified via immunofluorescence staining and Western blotting.

miR-196a overexpression significantly enhanced human CEC viability, promoted cell cycle progression, and upregulated stemness markers, while its inhibition produced opposite effects. Crucially, miR-196a increased mitochondrial membrane potential and suppressed markers associated with mitophagy, indicating improved mitochondrial health. The microRNA also modulated autophagy, cytoskeletal dynamics, and inflammation-related pathways by regulating key proteins such as ROCK1, ROCK2, and PAD4. Furthermore, miR-196a activated prosurvival pathways, specifically YAP and MAPK, and reduced levels of the tumor suppressor p53.