Inhibition of microRNA-21-5p reduces keloid fibroblast autophagy and migration by targeting PTEN after electron beam irradiation

Abstract

Electron beam (EB) irradiation is useful to reduce the recurrence of keloids; however, the underlying mechanism remains unknown. MicroRNA-21 (miR-21), which regulates autophagy during cancer radiation therapy, was identified as a potential therapeutic target for keloids. Here, we investigate the regulatory mechanism(s) of miR-21-5p on keloid fibroblast autophagy and migration after EB irradiation. The microRNA expression profile of the keloid dermis was examined by performing a microRNA microarray. Levels of LC3B and Beclin-1 were detected by immunohistochemical and western blot analysis in the keloid dermis and fibroblasts. Autophagy and apoptosis were tested in keloid fibroblasts after EB irradiation or transfection with an miR-21-5p inhibitor using electron microscopy, a Cyto-ID Green Autophagy Detection Kit, and an Annexin V PE Apoptosis Detection Kit. Migration was analyzed by an in vitro scratch–wound healing assay. Mechanistic tests were performed using small interfering RNAs to phosphatase and tensin homolog (siPTEN). Levels of miR-21-5p, PTEN, programmed cell death 4 (PDCD4), p-AKT, and apoptosis- and autophagy-associated genes were examined by qRT-PCR and western blotting. LC3B expression and migration ability were enhanced in fibroblasts and the keloid margin dermis compared with those in the adjacent normal skin. Both EB irradiation and an miR-21-5p inhibitor reduced keloid fibroblast autophagy, which was accompanied by decreased expression of miR-21-5p, p-AKT, and LC3B-II and increased expression of PTEN, PDCD4, and apoptosis-related genes. MiR-21-5p downregulation inhibited migration and suppressed LC3B expression and this was reversed by PTEN reduction. In conclusion, with increasing apoptosis, EB irradiation inhibits autophagy in keloid fibroblasts by reducing miR-21-5p, which regulates migration and LC3B expression via PTEN/AKT signaling. These data suggest a potential mechanism wherein miR-21-5p inhibition regulates autophagy and migration in EB-irradiated keloid fibroblasts, effectively preventing local invasion and recurrence. Therefore, miR-21-5p could be a new therapeutic target, to replace EB irradiation, and control keloid relapse.