RNA-seq Analysis For Exploring The Pathogenesis Of Retinitis Pigmentosa Caused By Rhodopsin P23H Mutation

Introduction: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 ER stress,pyroptosis,oxidative stress,calpain and glia cells activation 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.Methods:As C57BL/6J mice served as the control,RhoP33H/P23H mice at postnatal day 07(PN07),postnatal day 10(PN10),postnatal day 14(PN14),postnatal day 21(PN21)and postnatal day 35(PN35)were used for HE staining and electroretinogram to evaluate the structure and function of the retina.Total RNA at PN07,PN14,PN21 and PN35 were used for RNA sequencing.Differential genes expression analysis,GO,and KEGG functional enrichment analyses were conducted for RNA-seq data.Additionally,data of CRISPR screening libraries and the RNA-seq,which from reported researches,were used for generating custom gene sets for gene set enrichment analysis(GSEA).Next,we obtained the intersection of the above gene sets and our RNA-seq data to identify candidate genes,which were verified through real-time quantitative PCR(qPCR)and Western Blot(WB).Results:Functional enrichment analysis were consistent with disease phenotypes.In the results of GSEA,all time points excepted PN07 observed pyroptosis;ER stress,pyroptosis and oxidative stress were observed at PN14;ER stress and pyroptosis were shown in PN35.A total of 22 candidate genes were identified.The expression levels of selected genes(Ifi44,Kcnj14 and C4b)verified by qPCR were concordant with the RNA-seq data.And the mRNA levels of the three genes were significantly different between C57BL/6J and RhoP23H/P23H mice.The protein levels of Ifi44 and Kcnj14 were verified by WB.The change of IFI44 protein level was in accordance with the disease progression,but there was no significant difference in KCNJ14 protein level between the two groups.Conclusions: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(Ifi44,Kcnj14 and C4b)related to ER stress and pyroptosis,which deserve further study.These findings provide a novel perspective for the investigation of P23H-related adRP.