FTO-mediated m6A demethylation of KLF4 promotes the proliferation and collagen deposition of keloid fibroblasts

This study aims to elucidate the molecular mechanism mechanism by which FTO affects fibroblast proliferation and collagen deposition in keloids. Human keloid fibroblasts (KFs) and normal fibroblasts were cultured in vitro. FTO expression was silenced in KFs, and cell viability and proliferation were evaluated via CCK-8 and clone formation assays. FTO, KLF4, and MC1R expressions were quantified via qRT-PCR, while the protein levels of FTO, KLF4, MC1R, Collagen I, and Collagen III were determined by Western blot. The m⁶A RNA methylation status of total RNA was evaluated using the EpiQuik m6A RNA Methylation Quantification Kit. Post-actinomycin D treatment, the stability of KLF4 mRNA and its m⁶A modification level were measured. ChIP and dual-luciferase reporter assays confirmed the binding between KLF4 and MC1R promoter. KFs presented with significantly enhanced proliferation and collagen deposition, correlating with elevated FTO expression. Silence of FTO repressed the proliferation and collagen deposition of KFs, and elevated the m6A levels of total RNA and KLF4 mRNA in KFs, resulting in enhanced KLF4 mRNA stability and expression. KLF4 bound to the MC1R promoter and promoted MC1R expression. In conclusion, FTO represses KLF4 expression by removing m6A modification and further diminishes MC1R expression, thereby facilitating KF proliferation and collagen deposition.