RNA sequencing identified 475 differentially expressed genes (DEGs) in the TGF-β1-induced primary lung fibrosis model. Gene expression chips GSE101286 and GSE110147 from NCBI gene expression omnibus (GEO) database were analyzed using GEO2R, revealing 94 DEGs in IPF lung tissue samples. The gene ontology (GO) and pathway enrichment, Protein-protein interaction (PPI) network construction, and Maximal Clique Centrality (MCC) scoring were performed. Experimental validation included RT-qPCR, Immunohistochemistry (IHC), and Western Blot, with siRNA used for gene knockdown. A co-expression network was constructed by GeneMANIA.
GO enrichment highlighted significant enrichment of DEGs in TGF-β cellular response, connective tissue development, extracellular matrix components, and signaling pathways such as the AGE-RAGE signaling pathway and ECM-receptor interaction. PPI network analysis identified hub genes, including FN1, COL1A1, POSTN, KIF11, and ECT2. CALD1 (Caldesmon 1), CDH2 (Cadherin 2), and POSTN (Periostin) were identified as dysregulated hub genes in both the RNA sequencing and GEO datasets. Validation experiments confirmed the upregulation of CALD1, CDH2, and POSTN in TGF-β1-treated fibroblasts and IPF lung tissue samples. IHC experiments probed tissue-level expression patterns of these three molecules. Knockdown of CALD1, CDH2, and POSTN attenuated the expression of fibrotic markers (collagen I and α-SMA) in response to TGF-β1 stimulation in primary fibroblasts. Co-expression analysis revealed interactions between hub genes and predicted genes involved in actin cytoskeleton regulation and cell-cell junction organization.
CALD1, CDH2, and POSTN, identified as potential contributors to pulmonary fibrosis, present promising therapeutic targets for IPF patients.
■RNA测序鉴定了TGF-β1诱导的原发性肺纤维化模型中的475个差异表达基因(DEGs)。使用GEO2R分析来自NCBI基因表达综合(GEO)数据库的基因表达芯片GSE101286和GSE110147,揭示IPF肺组织样本中的94个DEG。基因本体论(GO)和途径富集,蛋白质-蛋白质相互作用(PPI)网络构建,并进行最大集团中心性(MCC)评分。实验验证包括RT-qPCR,免疫组织化学(IHC),和西方印迹,siRNA用于基因敲低。通过GeneMANIA构建共表达网络。
■GO富集强调了TGF-β细胞反应中DEGs的显着富集,结缔组织发育,细胞外基质成分,和信号通路,如AGE-RAGE信号通路和ECM-受体相互作用。PPI网络分析确定了枢纽基因,包括FN1、COL1A1、POSTN、KIF11和ECT2。CALD1(Caldesmon1),CDH2(钙黏着蛋白2),和POSTN(Periostin)在RNA测序和GEO数据集中都被鉴定为异常调节的hub基因。验证实验证实了在TGF-β1处理的成纤维细胞和IPF肺组织样品中CALD1、CDH2和POSTN的上调。IHC实验探测了这三种分子的组织水平表达模式。CALD1,CDH2和POSTN的敲除减弱了对原代成纤维细胞中TGF-β1刺激的纤维化标志物(胶原蛋白I和α-SMA)的表达。共表达分析揭示了hub基因和预测的参与肌动蛋白细胞骨架调节和细胞-细胞连接组织的基因之间的相互作用。
■CALD1、CDH2和POSTN,被确定为肺纤维化的潜在贡献者,为IPF患者提供了有希望的治疗目标。