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Molecular investigation of candidate genes for pyroptosis-induced inflammation in diabetic retinopathy
Nan Wang1,2,3, Lexi Ding1,2,3, Die Liu1,2,3
1Eye Center of Xiangya Hospital, Central South University, Changsha, China.
Background:
Diabetic retinopathy is a diabetic microvascular complication. Pyroptosis, as a way of inflammatory death, plays an important role in the occurrence and development of diabetic retinopathy, but its underlying mechanism has not been fully elucidated. The purpose of this study is to identify the potential pyroptosis-related genes in diabetic retinopathy by bioinformatics analysis and validation in a diabetic retinopathy model and predict the microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) interacting with them. Subsequently, the competing endogenous RNA (ceRNA) regulatory network is structured to explore their potential molecular mechanism.
Methods:
We obtained mRNA expression profile dataset GSE60436 from the Gene Expression Omnibus (GEO) database and collected 51 pyroptosis-related genes from the PubMmed database. The differentially expressed pyroptosis-related genes were obtained by bioinformatics analysis with R software, and then eight key genes of interest were identified by correlation analysis, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and protein-protein interaction (PPI) network analysis. Then, the expression levels of these key pyroptosis-related genes were validated with quantitative real-time polymerase chain reaction (qRT-PCR) in human retinal endothelial cells with high glucose incubation, which was used as an in vitro model of diabetic retinopathy. Western blot was performed to measure the protein levels of gasdermin D (GSDMD), dasdermin E (GSDME) and cleaved caspase-3 in the cells. Moreover, the aforementioned genes were further confirmed with the validation set. Finally, the ceRNA regulatory network was structured, and the miRNAs and lncRNAs which interacted with CASP3, TLR4, and GBP2 were predicted.
Results:
A total of 13 differentially expressed pyroptosis-related genes were screened from six proliferative diabetic retinopathy patients and three RNA samples from human retinas, including one downregulated gene and 12 upregulated genes. A correlation analysis showed that there was a correlation among these genes. Then, KEGG pathway and GO enrichment analyses were performed to explore the functional roles of these genes. The results showed that the mRNA of these genes was mainly related to inflammasome complex, interleukin-1 beta production, and NOD-like receptor signaling pathway. In addition, eight hub genes-CASP3, TLR4, NLRP3, GBP2, CASP1, CASP4, PYCARD, and GBP1-were identified by PPI network analysis using Cytoscape software. High glucose increased the protein level of GSDMD and GSDME, as critical effectors of pyroptosis, in retinal vascular endothelial cells. Verified by qRT-PCR, the expression of all these eight hub genes in the in vitro model of diabetic retinopathy was consistent with the results of the bioinformatics analysis of mRNA chip. Among them, CASP4, GBP1, CASP3, TLR4, and GBP2 were further validated in the GSE179568 dataset. Finally, 20 miRNAs were predicted to target three key genes-CASP3, GBP2, and TLR4, and 22 lncRNAs were predicted to potentially bind to these 20 miRNAs. Then, we constructed a key ceRNA network that is expected to mediate cellular pyroptosis in diabetic retinopathy.
Conclusion:
Through the data analysis of the GEO database by R software and verification by qRT-PCR and validation set, we successfully identified potential pyroptosis-related genes involved in the occurrence of diabetic retinopathy. The key ceRNA regulatory network associated with these genes was structured. These findings might improve the understanding of molecular mechanisms underlying pyroptosis in diabetic retinopathy.
Insights
This study identifies key pyroptosis-related genes in diabetic retinopathy using bioinformatics and experimental validation. A competing endogenous RNA (ceRNA) network was constructed to elucidate the molecular mechanisms of pyroptosis in diabetic retinopathy.
Area of Science:
- Ophthalmology
- Molecular Biology
- Genetics
Background:
- Diabetic retinopathy (DR) is a microvascular complication of diabetes.
- Pyroptosis, an inflammatory cell death pathway, contributes to DR pathogenesis.
- The precise molecular mechanisms of pyroptosis in DR remain unclear.
Purpose of the Study:
- To identify pyroptosis-related genes implicated in diabetic retinopathy.
- To validate these genes in an in vitro DR model.
- To construct a competing endogenous RNA (ceRNA) network for mechanistic insights.
Main Methods:
- Bioinformatic analysis of Gene Expression Omnibus (GEO) dataset GSE60436.
- Identification of differentially expressed pyroptosis-related genes using R software.
- Validation of key genes via qRT-PCR and Western blot in high-glucose treated retinal endothelial cells.
- Construction of a ceRNA network involving microRNAs (miRNAs) and long non-coding RNAs (lncRNAs).
Main Results:
- Thirteen differentially expressed pyroptosis-related genes were identified, with 12 upregulated.
- Eight hub genes (CASP3, TLR4, NLRP3, GBP2, CASP1, CASP4, PYCARD, GBP1) were pinpointed.
- High glucose increased pyroptosis effector protein levels (GSDMD, GSDME).
- A ceRNA network involving miRNAs and lncRNAs targeting CASP3, GBP2, and TLR4 was constructed.
Conclusions:
- Key pyroptosis-related genes and their regulatory network in diabetic retinopathy were successfully identified.
- The findings provide a deeper understanding of pyroptosis's molecular mechanisms in DR.
- This research may offer potential therapeutic targets for diabetic retinopathy.
