目的:内质网应激(ERS)与骨关节炎(OA)的发生密切相关,但是潜在的机制还没有完全描述。本研究旨在研究ERS相关基因在调节OA进展中的作用。
方法:从基因表达综合(GEO)数据库下载OA患者和正常人的表达谱。通过构建蛋白质-蛋白质相互作用(PPI)网络,通过R软件筛选和鉴定数据集GSE55457和GSE55235中的差异表达基因(DEGs)。通过STRING和Venn图分析,获得了hubERS相关基因。进行了基因本体论(GO)和基因和基因组的京都百科全书(KEGG)富集分析。研究了对骨关节炎(OA)具有高诊断价值的生物标志物。应用苏木精-伊红(H&E)染色和micro-CT评价OA模型的建立。使用逆转录定量聚合酶链反应(RT-qPCR)和蛋白质印迹验证了生物标志物的表达水平。最后,我们通过CIBERSORT算法评估了中心ERS相关基因与免疫浸润细胞的相关性.
结果:共鉴定出60个下调和52个上调的DEG,以下GO和KEGG通路分析证实这些DEGs主要富集在生物过程(BP)中,细胞成分(CC),分子函数(MF),和炎症相关的信号通路。有趣的是,在所有的DEG中,六个内质网应激相关基因,包括激活转录因子3(ATF3),DEAD-Box螺旋酶3X链接(DDX3X),AP-1转录因子亚基(JUN),真核起始因子4(EIF4A1),KDEL内质网蛋白滞留受体3(KDELR3),和血管内皮生长因子A(VEGFA),被发现与OA进展密切相关,随后的RT-qPCR和Western印迹分析证实DDX3X,JUN,VEGFA上调,而KDELR3,EIF4A1和ATF3在OA大鼠组织中与正常组织相比下调,这符合我们的生物信息学研究结果。此外,我们的受试者工作特征(ROC)曲线分析证实,上述6种ER应激相关基因可作为OA诊断的理想生物标志物,这些基因还可能通过影响肥大细胞和巨噬细胞的生物学功能来调节免疫反应.
结论:总的来说,本研究首次确定了六个内质网应激相关基因(ATF3、DDX3X、JUN,EIF4A1,KDELR3和VEGFA)可能在调节OA的进展中起关键作用。
OBJECTIVE: Endoplasmic reticulum stress (ERS) has been reported to be closely associated with the development of osteoarthritis (OA), but the underlying mechanisms are not fully delineated. The present study was designed to investigate the involvement of ERS-related genes in regulating OA progression.
METHODS: The expression profiles of OA patients and normal people were downloaded from the gene expression omnibus (GEO) database. The differentially expressed genes (DEGs) in datasets GSE55457 and GSE55235 were screened and identified by R software with the construction of the protein-protein interaction (PPI) networks. Through the STRING and Venn diagram analysis, hub ERS-related genes were obtained. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed. Biomarkers with high diagnostic values of osteoarthritis (OA) were studied. The hematoxylin and eosin (H&E) staining and micro-CT were applied to evaluate the establishment of the OA model. The expression levels of biomarkers were validated with the use of reverse transcription‑quantitative polymerase chain reaction (RT-qPCR) and western blot. Finally, we evaluated the correlations of hub ERS-related genes with the immune infiltration cells via the CIBERSORT algorithm.
RESULTS: A total of 60 downregulated and 52 upregulated DEGs were identified, and the following GO and KEGG pathway analyses verified that those DEGs were mainly enriched in biological process (BP), cellular component (CC), molecular function (MF), and inflammation-associated signal pathways. Interestingly, among all the DEGs, six ER stress-associated genes, including activating transcription factor 3 (ATF3), DEAD-Box Helicase 3 X-Linked (DDX3X), AP-1 transcription factor subunit (JUN), eukaryotic initiation factor 4 (EIF4A1), KDEL endoplasmic reticulum protein retention receptor 3 (KDELR3), and vascular endothelial growth factor A (VEGFA), were found to be closely associated with OA progression, and the following RT-qPCR and Western Blot analysis confirmed that DDX3X, JUN, and VEGFA were upregulated, whereas KDELR3, EIF4A1, and ATF3 were downregulated in OA rats tissues compared to the normal tissues, which were in accordance with our bioinformatics findings. Furthermore, our receiver operating characteristic (ROC) curve analysis verified that the above six ER stress-associated genes could be used as ideal biomarkers for OA diagnosis and those genes also potentially regulated immune responses by influencing the biological functions of mast cells and macrophages.
CONCLUSIONS: Collectively, the present study firstly identified six ER stress-associated genes (ATF3, DDX3X, JUN, EIF4A1, KDELR3, and VEGFA) that may play critical role in regulating the progression of OA.