miR-214 plays a major role in the self-renewal of skin tissue. However, whether miR-214 regulates the proliferation and differentiation of human hair follicle stem cells (HFSCs) is unknown. Primary HFSCs were isolated from human scalp skin tissue, cultured, and identified using flow cytometry. An miR-214 mimic and inhibitor were constructed for transfection into HFSCs. The MTS and colony formation assays examined cell proliferation. Immunofluorescence detected the localization and expression levels of TCF4, β-catenin, and differentiation markers. Luciferase reporter and TOP/FOP Flash assays investigated whether miR-214 targeted EZH2 and regulated the Wnt/β-catenin signaling pathway. Western blot determined the expression levels of enhancer of zeste homolog 2 (EZH2), Wnt/β-catenin signaling-related proteins, and HFSC differentiation markers in cells subjected to miR-214 transfection. miR-214 expression was remarkably decreased during the proliferation and differentiation of HFSCs into transit-amplifying (TA) cells. Downregulation of miR-214 promotes the proliferation and differentiation of HFSCs. Overexpression of miR-214 led to decreased expression of EZH2, β-catenin, and TCF-4, whereas downregulation of miR-214 resulted in increased expression of EZH2, β-catenin, and TCF-4 as well as TA differentiation markers. Immunofluorescence assay revealed that inhibiting miR-214 triggered the entry of β-catenin and TCF-4 into the nucleus. The luciferase reporter and TOP/FOP Flash assays demonstrated that miR-214 directly targets EZH2 and affects Wnt/β-catenin signaling. The miR-214/EZH2/β-catenin axis could be considered a candidate target in tissue engineering and regenerative medicine for HFSCs.
Keywords: EZH2; Hair follicles stem cells; Transit-amplifying cells; Wnt/β-catenin signal; miR-214.