Harry S. Truman Memorial Veterans' Hospital, Columbia, MO, USA; Department of Veterinary Medicine & Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
Harry S. Truman Memorial Veterans' Hospital, Columbia, MO, USA; Department of Veterinary Medicine & Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA; Department of Ophthalmology, School of Medicine, University of Missouri, Columbia, MO, USA.
Exp Eye Res. 2024 Nov;248:110073. doi: 10.1016/j.exer.2024.110073. Epub 2024 Sep 5.
This study analyzed the transcriptional changes in primary human corneal stromal fibroblasts (hCSFs) grown under quiescent (serum-free) and proliferating (serum-supplemented) culture conditions to identify genes, pathways, and protein‒protein interaction networks influencing corneal repair and regeneration. Primary hCSFs were isolated from donor human corneas and maintained in serum-free or serum-laden conditions. RNA was extracted from confluent cultures using Qiagen kit and subjected to RNA sequencing (RNAseq) analysis. Differential gene expression (DGE) and pathway enrichment analyses were conducted using DESeq2 and Gene Set Enrichment Analysis (GSEA), respectively. Protein‒protein interaction (PPI) networks were created exploiting the STRING database and analyzed with Cytoscape and the cytoHubba plugin. RNA-seq revealed 5,181 genes that were significantly differentially expressed/changed among the 18,812 annotated genes (p value ˂0.05). A cutoff value of a log2-fold change of ±1.5 or greater was used to identify 674 significantly upregulated and 771 downregulated genes between quiescent and proliferating hCSFs. Pathway enrichment analysis revealed significant changes in genes linked to cell cycle regulation, inflammatory, and oxidative stress response pathways, such as E2F Targets, G2M Checkpoint, and MYC Targets, TNFA signaling via NF-kB, and oxidative phosphorylation. Protein-protein interaction network analysis highlighted critical hub genes. The FGF22, CD34, ASPN, DPT, LUM, FGF10, PDGFRB, ECM2, DCN, VEGFD, OMD, OGN, ANGPT1, CDH5, and PRELP were upregulated, whereas genes linked to cell cycle regulation and mitotic progression, such as BUB1, TTK, KIF23, KIF11, BUB1B, DLGAP5, NUSAP1, CCNA2, CCNB1, BIRC5, CDK1, KIF20A, AURKB, KIF2C, and CDCA8, were downregulated. The RNA sequences and gene count files have been submitted to the Gene Expression Omnibus (accession # GSE260476). Our study provides a comprehensive information on the transcriptional and molecular changes in hCSFs under quiescent and proliferative conditions and highlights key pathways and hub genes.
本研究分析了在静止（无血清）和增殖（补充血清）培养条件下生长的原代人角膜基质成纤维细胞（hCSFs）的转录变化，以鉴定影响角膜修复和再生的基因、途径和蛋白质-蛋白质相互作用网络。从供体人角膜中分离原代 hCSFs，并在无血清或富含血清的条件下维持。使用 Qiagen 试剂盒从汇合培养物中提取 RNA，并进行 RNA 测序（RNAseq）分析。使用 DESeq2 和基因集富集分析（GSEA）分别进行差异基因表达（DGE）和途径富集分析。利用 STRING 数据库创建蛋白质-蛋白质相互作用（PPI）网络，并利用 Cytoscape 和 cytoHubba 插件进行分析。RNA-seq 显示在 18812 个注释基因中，有 5181 个基因的表达/变化显著不同（p 值<0.05）。使用对数倍变化的 ±1.5 或更大的截断值来识别静止和增殖 hCSFs 之间 674 个显著上调和 771 个下调基因。途径富集分析显示，与细胞周期调控、炎症和氧化应激反应途径相关的基因发生了显著变化，如 E2F 靶标、G2M 检查点和 MYC 靶标、TNFA 信号通过 NF-kB 和氧化磷酸化。蛋白质-蛋白质相互作用网络分析突出了关键的枢纽基因。FGF22、CD34、ASPN、DPT、LUM、FGF10、PDGFRB、ECM2、DCN、VEGFD、OMD、OGN、ANGPT1、CDH5 和 PRELP 上调，而与细胞周期调控和有丝分裂进展相关的基因，如 BUB1、TTK、KIF23、KIF11、BUB1B、DLGAP5、NUSAP1、CCNA2、CCNB1、BIRC5、CDK1、KIF20A、AURKB、KIF2C 和 CDCA8，下调。RNA 序列和基因计数文件已提交给基因表达综合数据库（注册号#GSE260476）。本研究提供了静止和增殖条件下 hCSFs 转录和分子变化的全面信息，并强调了关键途径和枢纽基因。
