Novel microRNA seq-14465_x69 promotes scarless wound healing by targeting TGF-β/SMADs pathway in keratinocytes

Fetal skin in early to mid gestation exhibits scar-free wound healing, however, it loses the regenerative capacity in late gestation and adulthood, where fibrosis predominates. Human keratinocytes (hKCs) and human fibroblasts (hFBs) play critical roles in this process, with early- to mid- gestational hKCs enhancing hFB proliferation and migration. MicroRNAs (miRNAs) regulate wound healing, but their roles in scarless repair remains incompletely disclosed. We isolated hKCs from mid- (22-23 weeks) and late- gestational (33-36 weeks) fetal skin and performed miRNA sequencing (miRNA-seq), identifying seq-14465_x69 as a novel miRNA highly expressed in mid gestation. Bioinformatics and dual-luciferase reporter assays predicted and validated its targeting of TGF-β/SMADs pathway genes. Functional assays including cell proliferation, migration, and a murine full-thickness wound model determined its effects on proliferation, migration, and fibrosis during wound healing. Seq-14465_x69 suppressed TGF-β2, TGF-βR2, and SMAD3, enhanced hFBs migration while reducing collagen I deposition, and drove accelerated wound closure. Our study revealed seq-14465_x69 as a key miRNA promoting scarless healing by modulating TGF-β/SMADs signaling and extracellular matrix (ECM) remodeling, offering a potential therapeutic target for anti-fibrotic wound management.