Therapeutic Potential of miR-196a in Protecting Corneal Endothelial Cells From Senescence Through Mitochondrial and TGF-β Signaling Modulation

Purpose: Corneal endothelial cells (CECs) play a vital role in maintaining corneal transparency and function, with their hexagonal morphology being a hallmark of proper cellular packing and barrier integrity. This study investigates the functional role of miR-196a in regulating key cellular processes, including viability, cell cycle progression, stemness, mitochondrial function, and senescence. Methods: Human CECs were cultured and transfected with miR-196a mimics or inhibitors. Cell viability, proliferation, and cell cycle progression were assessed using the CCK-8 assay and flow cytometry. Gene expression was analyzed through real-time polymerase chain reaction and Western blotting. Mitochondrial function was evaluated using JC-1 staining and by measuring mitophagy markers. Senescence was induced using transforming growth factor β (TGF-β) and detected by senescence-associated β-galactosidase staining. Cytoskeletal changes were visualized through immunofluorescence staining. Signaling pathway activation was assessed using Western blotting and immunofluorescence. Results: miR-196a overexpression enhanced cell viability, promoted cell cycle progression, and upregulated stemness markers, while its inhibition had opposite effects. It increased mitochondrial membrane potential and suppressed mitophagy markers. miR-196a modulated autophagy, cytoskeletal dynamics, and inflammation-related pathways by regulating ROCK1, ROCK2, and PAD4. It activated prosurvival pathways (YAP, MAPK) and reduced p53 levels. Importantly, miR-196a mitigated TGF-β-induced senescence by improving mitochondrial function, suppressing autophagy, and modulating TGF-β signaling components. Conclusions: These results showed the protective role of miR-196a in maintaining cellular function and suggest its therapeutic potential for addressing aging-related diseases, fibrotic disorders, and mitochondrial dysfunction. This study underscores miR-196a as a promising target for therapies aimed at counteracting cellular senescence and promoting regenerative capacities. Translational Relevance: miR-196a may serve as a novel therapeutic target for preserving corneal endothelial function and preventing age-related degeneration.