■基质中角膜成纤维细胞向肌成纤维细胞的转分化是角膜伤口愈合中的中心机制事件。这项研究旨在使用RNA测序(RNA-seq)表征影响人角膜成纤维细胞(hCSF)转分化为人角膜肌成纤维细胞(hCMFs)的基因和途径,以开发全面的机制信息并确定角膜纤维化管理的新目标。
■初级hCSF来源于供体人角膜。通过在无血清条件下用转化生长因子β1(TGFβ1;5ng/mL)处理原代hCSF72小时来产生hCMF。使用RNeasyPlusMini试剂盒提取RNA,并在质量控制测试后进行RNA-seq分析。差异基因表达,途径富集,和蛋白质-蛋白质网络分析使用DESeq2,GSEA/PANTHER/Reactome,和Cytoscape/cytoHubba,分别。
■hCMFs和hCSFs的RNA-seq分析鉴定出3843个差异表达的基因和转录本(调整后的P<0.05)。log(倍数变化)≥±1.5过滤器显示两种细胞类型之间的816个上调和739个下调基因。通路富集分析显示上皮-间质转化的归一化富集评分最高(5.569),其次是mTORC1信号(2.949),血管生成(2.176),和TGFβ信号(2.008)。蛋白质-蛋白质相互作用网络分析确定了影响角膜肌成纤维细胞发育的前20个节点。通过实时定量逆转录PCR和免疫荧光验证了新型MXRA5在角膜基质中的表达及其与角膜纤维化的关系。将RNA-seq和基因计数文件提交至NCBI基因表达Omnibus(GSE260476)。
■这项研究发现了几个参与肌成纤维细胞发育的新基因,为开发角膜纤维化的新治疗策略提供了潜在的目标。
UNASSIGNED: Transdifferentiation of corneal fibroblasts to myofibroblasts in the stroma is a central mechanistic event in corneal wound healing. This study sought to characterize genes and pathways influencing transdifferentiation of human corneal fibroblasts (hCSFs) to human corneal myofibroblasts (hCMFs) using RNA sequencing (RNA-seq) to develop comprehensive mechanistic information and identify newer targets for corneal fibrosis management.
UNASSIGNED: Primary hCSFs were derived from donor human corneas. hCMFs were generated by treating primary hCSFs with transforming growth factor β1 (TGFβ1; 5 ng/mL) for 72 hours under serum-free conditions. RNA was extracted using the RNeasy Plus Mini Kit and subjected to RNA-seq analysis after quality control testing. Differential gene expression, pathway enrichment, and protein-protein network analyses were performed using DESeq2, GSEA/PANTHER/Reactome, and Cytoscape/cytoHubba, respectively.
UNASSIGNED: RNA-seq analysis of hCMFs and hCSFs identified 3843 differentially expressed genes and transcripts (adjusted P < 0.05). The log(fold change) ≥ ±1.5 filter showed 816 upregulated and 739 downregulated genes between two cell types. Pathway enrichment analysis showed the highest normalized enrichment score for epithelial-to-mesenchymal transition (5.569), followed by mTORC1 signaling (2.949), angiogenesis (2.176), and TGFβ signaling (2.008). Protein-protein interaction network analysis identified the top 20 nodes influencing corneal myofibroblast development. The expression of a novel MXRA5 in corneal stroma and its association with corneal fibrosis was verified by real-time quantitative reverse transcription PCR and immunofluorescence. RNA-seq and gene count files were submitted to the NCBI Gene Expression Omnibus (GSE260476).
UNASSIGNED: This study identified several novel genes involved in myofibroblast development, offering potential targets for developing newer therapeutic strategies for corneal fibrosis.