Proteomic analysis of TCF4 knock-out corneal endothelial cells derived from the patients with Fuchs endothelial corneal dystrophy

Abstract

Purpose : Patients with Fuchs endothelial corneal dystrophy (FECD) often harbor expansion of a trinucleotide CTG repeat in the TCF4 gene, thus suggesting that TCF4 is a potential causative gene for FECD. We previously reported that the expression of TCF4 is upregulated in the corneal endothelium of FECD patients. The purpose of this present study was to elucidate the involvement of TCF4 in the pathophysiology of FECD via proteomic analysis of an FECD cell model.

Methods : Corneal endothelial cells (CECs) isolated from FECD patients were immortalized using both SV40 and hTERT to produce an immortalized FECD cellular model (iFECD). TCF4 was knocked out using CRISPR/Cas9 in iFECD (TCF4-/- iFECD). Total proteins were isolated, detected by mass spectrometry, and analyzed by use of the SEQUEST^™ search algorithm through Proteome Discoverer software against Human RefSeq protein database. For functional enrichment analysis, the gene ontology (GO), KEGG, and Reactome pathways were investigated. In addition, the GeneMANIA Cytoscape plug-in was used to clarify protein-protein interaction (PPI) and hub proteins, which are involved in the knockout of TCF4.

Results : Ninety differentially expressed proteins (DEPs) (|log₂ fold change| > 0.5 and P value < 0.05) were identified among a total of 6510 identified proteins, including 52 upregulated and 38 downregulated proteins. GO pathway analysis revealed enrichment of extracellular matrix (ECM) organization, response to oxidative stress, and cell motility, KEGG pathway analysis revealed enrichment of ECM-receptor interaction, and Reactome pathway analysis demonstrated that multiple proteins related to collagen, integrin, and glycosaminoglycan were downregulated by loss of TCF4. Finally, GeneMANIA identified 20 hub proteins and showed that 5 of those 20 proteins were related to ECM.

Conclusions : Functional and pathway enrichment analyses revealed that TCF4 is involved in ECM-related pathways in the corneal endothelium of FECD. Our findings indicate that TCF4 plays an important role in the pathophysiology of FECD, probably by inducing excessive production of ECM proteins.

This is a 2021 ARVO Annual Meeting abstract.