miR-129-5p Modulates the ZEB1/2 Signaling Axis to Suppress Palmitic Acid-Induced Epithelial-Mesenchymal Transition...

Proliferative vitreoretinopathy (PVR) is driven by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. While palmitic acid (PA) represents a potent metabolic stressor in the subretinal microenvironment, its impact on the microRNA (miRNA) landscape remains poorly defined. This study investigated the role of miR-129-5p in PA-induced transdifferentiation and evaluated the protective potential of ectopic miR-129-5p mimicry in ARPE-19 cells.

Low-passage ARPE-19 cells were challenged with sublethal PA to induce lipotoxic stress. miR-129-5p levels were modulated using synthetic mimics under basal and stressed conditions. EMT progression was tracked using immunofluorescence for tight junction topology and transcription factor nuclear localization, Phalloidin-FITC cytoskeletal F-actin staining, and immunoblotting for hallmark epithelial (E-cadherin) and mesenchymal (α-smooth muscle actin, fibronectin) effectors. Functional shifts were evaluated via wound healing and paracellular macromolecular permeability assays.

PA exposure triggered a myofibroblastic phenotype and significantly depleted the intracellular miR-129-5p pool, accompanied by parallel vesicle-independent extracellular efflux. Under unchallenged baseline, mimic delivery directly suppressed endogenous ZEB1/2 expression. Under lipid stress, miR-129-5p mimicry neutralized transdifferentiation, successfully restoring E-cadherin and counteracting core transcription factor upregulation (ZEB1, ZEB2, and Snail). Morphologically, mimicry prevented pericellular ZO-1 dissolution, suppressed ZEB2 nuclear translocation, and blocked contractile stress fiber assembly. Functionally, maintaining this miRNA node significantly attenuated PA-enhanced cell migration and rescued outer blood-retinal barrier homeostasis by suppressing paracellular macromolecular flux.

miR-129-5p functions as an essential cell-autonomous posttranscriptional gatekeeper of RPE identity, cytoskeletal architecture, and barrier homeostasis. Targeted modulation of this posttranscriptional network offers a promising pharmacological framework for mitigating lipotoxicity-associated subretinal fibrosis in PVR.