CRYAA activates the SIRT1-pi3K/AKT signaling pathway by suppressing mir-155-5p to protect the RPE

Abstract

Background: Retinal degenerative diseases are important causes of blindness, and their pathogenesis is related to degenerative changes in the retinal pigment epithelium (RPE). αA-Crystallin (CRYAA) plays a role in maintaining cellular protein homeostasis and has been shown to protect the retina from stress; however, the detailed mechanisms involved in this protection are not known.

Methods: In vitro, ARPE-19 cells stably overexpressing CRYAA were generated from CRYAA-RPE cells. The effects of CRYAA overexpression on H₂O₂-induced RPE cell apoptosis were assessed via CCK-8 assays, flow cytometry, and reactive oxygen species (ROS) quantification. Differences in miR-155-5p levels between RPE and CRYAA-RPE cells were determined via RT‒qPCR. SIRT1 was predicted as a downstream target gene of miR-155-5p, and the relative luciferase activities of NC, miR-155-5p mimic with SIRT1 WT 3'-UTR and SIRT1 MT 3'-UTR reporter plasmids were determined using dual luciferase gene reporter assays. The expression of PI3K/AKT signaling pathway-related proteins was assessed by Western blotting. For in vivo experiments, a mouse model of retinal degeneration was constructed with sodium iodate, and the extent of retinal damage was assessed via histopathological analysis.

Results: In vitro experiments revealed that CRYAA overexpression significantly reduced apoptosis and decreased ROS levels as well as miR-155-5p expression. Additionally, the specific binding site of miR-155-5p to the SIRT1 3'-UTR was confirmed by bioinformatics prediction and a dual luciferase reporter assay. CRYAA overexpression increased SIRT1 expression, which further activated the PI3K/AKT signaling pathway, exerting a protective function. After the SIRT1 gene silencing or miR-155-5p overexpression, the PI3K/AKT signaling pathway was inhibited correspondingly. In vivo experiments revealed that vitreous Cryaa-AAV injection alleviated sodium iodate-induced retinal degeneration in mice, significantly improving retinal function.

Conclusions: CRYAA activates the PI3K/AKT signaling pathway by decreasing miR-155-5p expression and increasing SIRT1 level, which protects RPE cells from apoptosis. These findings provide a new approach for the treatment of retinal degenerative diseases.

Keywords: Apoptosis; PI3K/AKT signaling pathway; Retinal pigment epithelium; SIRT1; miR-155–5p; αA-crystallin.