BACKGROUND: Keloid scarring is caused by a fibroproliferative disorder due to abnormal activation of genes, the underlying mechanism of which is still unclear. The basic helix-loop-helix transcription factor Twist-related protein 1 (TWIST1) controls cell proliferation and differentiation in tissue development and disease processes. In this study, we aimed to clarify the essential role of TWIST1 in the pathogenesis of keloids.
METHODS: 1.Single-Cell RNA Sequencing: Analyzed 28,064 cells from keloid and adjacent normal tissues to map cellular heterogeneity (n=4 patients).
2.Functional Assays: In vitro: CCK-8, Transwell, immunofluorescence, and Western blotting in KFBs treated with TWIST1 inhibitor harmine. Molecular Interactions: Co-immunoprecipitation (Co-IP), ubiquitination assays, chromatin immunoprecipitation (ChIP-qPCR), and dual-luciferase reporter assays to validate TWIST1-MEF2A-TBR1 interactions.
3.Pathway Analysis: Gene set enrichment (GSEA) for TGF-ß, Eph-ephrin, and tumor-related pathways.
RESULTS: 1.Cellular Heterogeneity: SCRNA-seq identified expanded fibroblast (cluster c9) and VEC subpopulations (clusters c4, c5, c18) in keloids, linked to TGF-ß and Eph-ephrin pathway activation. TWIST1 was significantly upregulated in KFBs and VECs (p<0.01).
2.TWIST1 Functional Roles: Fibrosis: TWIST1 promoted collagen synthesis (COL1A1, COL3A1) and myofibroblast activation via TGF-B/Smad3. Harmine (TWIST1 inhibitor) suppressed TWIST1, reducing ECM deposition (p<0.05).
Angiogenesis: TWIST1 enhanced Eph-ephrin signaling (EFNB2-EPHA4) in VECs, driving pathological vascularization.
3.Mechanistic Insights: TWIST1 stabilized MEF2A by inhibiting MDM2-mediated ubiquitination, prolonging its half-life. MEF2A directly bound the TBR1 promoter, enhancing TGF-ß receptor expression (ChIP-qPCR fold enrichment=2.5, p<0.001). TWIST1 overexpression rescued TBR1 expression in KFBs, while MEF2A knockdown reversed this effect (p <0.01).
CONCLUSION: Our research highlights a significant function of TWIST1 in the development of keloid and its related fibroblasts, partially facilitated by elevated MEF2A-dependent TBR1 expression.
Blocking the expression of TWIST1 in KFBs could potentially pave a novel therapeutic avenue for keloid treatment.
