Lv Jia, Kou Nannan, Li Yunxuan, Qiu Kejia, Guo Xiang, Zhang Li, Zhang Zhichao, He Shaoxuan, Yuan Yong
Department of Trauma Surgery, The Second Affiliated Hospital of Kunming Medical University, 374 Yunnan-Myanmar Avenue, Kunming, 650101, China.
Biochem Genet. 2024 May 11. doi: 10.1007/s10528-024-10818-1.
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.
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.
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.
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.
内质网应激（ERS）已被报道与骨关节炎（OA）的发展密切相关，但其潜在机制尚未完全阐明。本研究旨在探讨ERS相关基因在调节OA进展中的作用。
从基因表达综合数据库（GEO）下载OA患者和正常人的表达谱。使用R软件筛选并鉴定数据集GSE55457和GSE55235中的差异表达基因（DEG），并构建蛋白质-蛋白质相互作用（PPI）网络。通过STRING和维恩图分析，获得核心ERS相关基因。进行基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析。研究具有高骨关节炎（OA）诊断价值的生物标志物。应用苏木精-伊红（H&E）染色和显微CT评估OA模型的建立。使用逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法验证生物标志物的表达水平。最后，通过CIBERSORT算法评估核心ERS相关基因与免疫浸润细胞的相关性。
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DEG主要富集在生物过程（BP）、细胞成分（CC）、分子功能（MF）和炎症相关信号通路中。有趣的是，在所有DEG中，发现六个内质网应激相关基因，包括激活转录因子3（ATF3）、X连锁的DEAD盒解旋酶3（DDX3X）、AP-1转录因子亚基（JUN）、真核起始因子4（EIF4A1）、KDEL内质网蛋白保留受体3（KDELR3）和血管内皮生长因子A（VEGFA）与OA进展密切相关，随后的RT-qPCR和蛋白质免疫印迹分析证实，与正常组织相比，OA大鼠组织中DDX3X、JUN和VEGFA上调，而KDELR3、EIF4A1和ATF3下调，这与我们的生物信息学结果一致。此外，我们的受试者工作特征（ROC）曲线分析证实，上述六个内质网应激相关基因可作为OA诊断的理想生物标志物，并且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。
总体而言，本研究首次鉴定出六个内质网应激相关基因（ATF3、DDX3X、JUN、EIF4A1、KDELR3和VEGFA），它们可能在调节OA进展中起关键作用。
