# Protective Effects of miR-16-5p and miR-142-3p on Inflammation and Autophagy in Human Corneal Epithelial Cells Under Hyperosmotic Stress In Vitro

## TL;DR

This study shows that miR-16-5p and miR-142-3p reduce inflammation and autophagy in corneal cells under hyperosmotic stress, suggesting their potential in treating dry eye disease.

## Contribution

The novel finding is that miR-16-5p and miR-142-3p regulate inflammation and autophagy in corneal epithelial cells under hyperosmotic stress.

## Key findings

- miR-16-5p and miR-142-3p reduce inflammation by suppressing NF-κB-mediated cytokine expression.
- Both miRNAs decrease oxidative stress and restore antioxidant defenses in hyperosmotic conditions.
- Overexpression of these miRNAs normalizes autophagy and apoptosis markers in corneal epithelial cells.

## Abstract

To investigate the regulatory effects of miR-16-5p and miR-142-3p on inflammation and autophagy in human corneal epithelial cells (HCEpiCs) exposed to hyperosmotic stress, a key pathogenic condition in dry eye disease, HCEpiCs were cultured under NaCl-induced hyperosmotic conditions (450 mOsm, 24 h) and transfected with miR-16-5p or miR-142-3p mimics. Expression of inflammatory cytokines (IL-1β, IL-6, TNF-α, IRAK1), autophagy-related genes (ATG5, Beclin-1, ATG16L1, p62), and apoptotic markers (Bax, Bcl-2, caspase-3) was analyzed by qRT-PCR and Western blot. Reactive oxygen species (ROS), autophagic vesicles, and apoptosis were evaluated using DCFH-DA, DAPRed, and Annexin V assays. The expression levels of antioxidant proteins (SOD1, catalase, NRF2) were also measured. Hyperosmotic stress induces marked inflammatory activation and excessive autophagy in HCEpiCs, accompanied by increased ROS generation and apoptosis. Overexpression of miR-16-5p or miR-142-3p significantly attenuated these effects by suppressing NF-κB-mediated cytokine expression and downregulating ATG5 and ATG16L1 expression, while restoring p62 expression. Both miRNAs reduced oxidative stress and COX-2 expression, enhanced antioxidant defenses, and normalized the expression of apoptotic markers. miR-16-5p and miR-142-3p are important regulators of inflammation and autophagy under hyperosmotic stress. Our findings suggest that modulating intracellular miR-16-5p and miR-142-3p levels in corneal epithelial cells may represent a potential approach to protect the ocular surface under hyperosmotic stress, although their systemic roles in autoimmune dry eye require further clarification.