Inhibiting miR-129-5p alleviates inflammation and modulates autophagy by targeting ATG14 in fungal keratitis

Exp Eye Res. 2021 Oct:211:108731. doi: 10.1016/j.exer.2021.108731. Epub 2021 Aug 17.

Abstract

To investigate the role of miR-129-5p in inflammation and autophagy in fungal keratitis, we established a keratitis mouse model infected with Fusarium solani (F. solani) and conducted experiments on corneal stromal cells infected with F. solani. The expression of miR-129-5p was detected via quantitative real-time polymerase chain reaction (PCR). The miR-129-5p antagomir was used to transfect cells and mice to study the regulatory role of miR-129-5p in autophagy and inflammation after fungal infection. The expression of Beclin1 and LC3B and colocalization of LC3B with lysosomes were detected via Western blotting and immunofluorescence. CCK-8 was used to determine the viability of corneal stromal cells. The expression of IL-1β were detected by ELISA. Bioinformatics software was used to predict the potential targets of miR-129-5p, which were verified by a luciferase reporter gene assay. RT-PCR showed that miR-129-5p expression in mouse corneas was significantly increased after infection with F. solani. Subconjunctival injection of the miR-129-5p antagomir significantly enhanced the proteins Beclin-1 and LC3B. At the same time, inhibiting miR-129-5p expression could reduce the inflammatory response in FK and significantly increase the viability of corneal stromal cells infected with F. solan. Moreover, the dual luciferase reporter assay indicated that Atg14 was a direct target of miR-129-5p. Our study shows that miR-129-5p is a novel small molecule that regulates autophagy by targeting Atg14, indicating that it may be a proinflammatory and therapeutic target for fungal keratitis.

Keywords: Atg14; Autophagy; Fungal keratitis; Fusarium solani; miR-129-5p.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antagomirs / pharmacology
  • Autophagy / drug effects*
  • Autophagy-Related Proteins / genetics*
  • Autophagy-Related Proteins / metabolism
  • Beclin-1 / metabolism
  • Blotting, Western
  • Corneal Ulcer / genetics
  • Corneal Ulcer / microbiology
  • Corneal Ulcer / prevention & control*
  • Disease Models, Animal
  • Eye Infections, Fungal / genetics
  • Eye Infections, Fungal / microbiology
  • Eye Infections, Fungal / prevention & control*
  • Fusariosis / genetics
  • Fusariosis / microbiology
  • Fusariosis / prevention & control*
  • Fusarium
  • Inflammation / prevention & control*
  • Lysosomes / metabolism
  • Mice
  • Mice, Inbred BALB C
  • MicroRNAs / antagonists & inhibitors*
  • MicroRNAs / physiology
  • Microscopy, Fluorescence
  • Microtubule-Associated Proteins / metabolism
  • Real-Time Polymerase Chain Reaction
  • Vesicular Transport Proteins / genetics*
  • Vesicular Transport Proteins / metabolism

Substances

  • Antagomirs
  • Atg14 protein, mouse
  • Autophagy-Related Proteins
  • Beclin-1
  • MIRN129 microRNA, mouse
  • Map1lc3b protein, mouse
  • MicroRNAs
  • Microtubule-Associated Proteins
  • Vesicular Transport Proteins

Supplementary concepts

  • Fusarium solani