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

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.