Transcriptomic landscape of quiescent and proliferating human corneal stromal fibroblasts

Abstract

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.

Keywords: Cornea; Fibroblasts; Proliferative; Quiescent; RNA sequencing; Stroma.