RNA-Seq Analysis for Exploring the Pathogenesis of Retinitis Pigmentosa in P23H Knock-In Mice

Mechanisms contributing to the progression of autosomal dominant retinitis pigmentosa (adRP) due to the P23H rhodopsin mutation are complex and diverse. Previous studies showed that mechanisms like endoplasmic reticulum (ER) stress, pyroptosis, and oxidative stress were involved in the pathogenesis of the disease. However, the roles and relationships of different mechanisms are not precisely known. In this study, we aimed to evaluate certain mechanisms and find novel genes involved in P23H-related adRP.

Total RNA extracted at postnatal day (PN) 14, PN21, and PN35 was used for RNA sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analyses were conducted for RNA-seq data. Additionally, data from the clustered regularly interspaced short palindromic repeats (CRISPR) screening library and the RNA-seq data of several mechanisms were used for generating custom gene sets for gene set enrichment analysis (GSEA). Next, we obtained the intersection of the aforementioned gene sets and our RNA-seq data to identify candidate genes, which were verified using real-time quantitative PCR (qPCR).

Functional enrichment analyses were consistent with disease phenotypes. All time points observed pyroptosis. In the results of GSEA, ER stress, pyroptosis, and oxidative stress were observed at PN14. ER stress and pyroptosis were shown on PN35. A total of 22 candidate genes were identified. The expression levels of selected genes verified by qPCR were concordant with the RNA-seq data.

In our study, we conclude that pyroptosis and ER stress might play a central role in RP progression. We also identified differentially expressed gene clusters related to ER stress and pyroptosis, which deserve further study. These findings provide a novel perspective for the investigation of P23H-related adRP.