Molecular investigation of candidate genes for pyroptosis-induced inflammation in diabetic retinopathy.
Molecular investigation of candidate genes for pyroptosis-induced inflammation in diabetic retinopathy.
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DOI：
10.3389/fendo.2022.918605
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发表时间：
2022
影响因子：
5.2
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中科院分区：
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糖尿病视网膜病变是一种糖尿病微血管并发症。焦亡作为炎症性死亡的一种方式，在糖尿病视网膜病变的发生发展中起着重要作用，但其潜在机制尚未完全阐明。本研究旨在通过生物信息学分析和糖尿病视网膜病变模型验证，识别糖尿病视网膜病变中潜在的焦热相关基因，并预测与之相互作用的microRNAs （miRNAs）和长链非编码rna （lncRNAs）。随后，构建竞争内源性RNA （ceRNA）调控网络，探索其潜在的分子机制。我们从Gene expression Omnibus （GEO）数据库中获得mRNA表达谱数据集GSE60436，并从PubMmed数据库中收集了51个与热解相关的基因。利用R软件进行生物信息学分析，获得差异表达的热解相关基因，然后通过相关性分析、基因本体（GO）富集分析、京都基因与基因组百科全书（KEGG）通路分析和蛋白-蛋白相互作用（PPI）网络分析，鉴定出8个感兴趣的关键基因。然后，在高糖培养的人视网膜内皮细胞中，采用定量实时聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）验证这些关键的焦热相关基因的表达水平，并将其作为糖尿病视网膜病变的体外模型。Western blot法检测细胞中dasdermin D （GSDMD）、dasdermin E （GSDME）和cleaved caspase-3的蛋白水平。并利用验证集对上述基因进行进一步确认。最后，构建了ceRNA调控网络，预测了与CASP3、TLR4和GBP2相互作用的mirna和lncrna。从6例增生性糖尿病视网膜病变患者和3例人视网膜RNA样本中，共筛选出13个差异表达的焦热相关基因，包括1个下调基因和12个上调基因。相关分析表明，这些基因之间存在相关性。然后，通过KEGG通路和GO富集分析来探索这些基因的功能作用。结果表明，这些基因的mRNA表达主要与炎性小体复合体、白细胞介素-1 β的产生以及nod样受体信号通路有关。此外，利用Cytoscape软件进行PPI网络分析，鉴定出8个枢纽基因——casp3、TLR4、NLRP3、GBP2、CASP1、CASP4、PYCARD和gbp1。高葡萄糖升高了视网膜血管内皮细胞中作为焦亡关键效应物的GSDMD和GSDME的蛋白水平。通过qRT-PCR验证，这8个枢纽基因在糖尿病视网膜病变体外模型中的表达与mRNA芯片生物信息学分析结果一致。其中，CASP4、GBP1、CASP3、TLR4、GBP2在GSE179568数据集中得到进一步验证。最后，预测了20个mirna靶向三个关键基因——casp3、GBP2和TLR4，并预测了22个lncrna可能与这20个mirna结合。然后，我们构建了一个关键的ceRNA网络，该网络有望介导糖尿病视网膜病变的细胞焦亡。通过R软件对GEO数据库进行数据分析，并通过qRT-PCR和验证集进行验证，我们成功鉴定出参与糖尿病视网膜病变发生的潜在焦热相关基因。构建了与这些基因相关的关键ceRNA调控网络。这些发现可能有助于了解糖尿病视网膜病变中焦亡的分子机制。
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. 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. 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. 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.
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