MiR-214 Regulates the Human Hair Follicle Stem Cell Proliferation and Differentiation by Targeting EZH2 and Wnt/β-Catenin Signaling Way In Vitro

Abstract

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.

Fig. 1

Primary HFSC culture and identification of HFSCs and miR-214 expression during proliferation and differentiation stages. A–F Primary HFSCs were successfully cultured and subcultured. A, B Day 15, HFSCs emerge from the tissue (10 × 10 and 10 × 20); C Day 21, HFSCs emerge from the tissue (10 × 10); D Day 7 after primary culture (scattered HFSCs, 10 × 10); E: 1st generation; F 2nd generation; G Identification of HFSCs by flow cytometry using LGR5 (+), CD200 (++), CD271 (−), and CK15 (−) as markers; H miR-214 expression during differentiation to TA cells

Fig. 2

Clone formation and MTS assays following miR-214 transfection. A Normal group; B Mock; C miR-214 mimic; D miR-214 inhibitor; E EZH2 siRNA; F XAV-939; G Plate clone formation statistics (*p < 0.05 vs. control, **p < 0.01 vs. control); H Proliferation rate; I Inhibition rate

Fig. 3

FACS analysis of the effect of altering miR-214 expression on cell cycle progression. A Normal group; B Mock; C miR-214 mimic; D miR-214 inhibitor; E EZH2 siRNA; F XAV-939; G Statistics of the cell cycle distribution

Fig. 4

Analysis of the miR-214 binding to the EZH2 3′-UTR by the luciferase activity assay. A miR-214 sequence with the 3′-UTRs of EZH2 in different species. The position of the miR-214 binding site on human EZH2 mRNA and the EZH2 3′-UTR mutant is depicted. B Addition of miR-214 mimic attenuated the EZH2 3′-UTR reporter gene activity. Mutation of the miR-214 putative target site blocked the suppressive effect of miR-214 on the target, suggesting EZH2 as a miR-214 target gene. p < 0.05 versus control mimic. C TOP Flash and FOP Flash assays assessed the effect of miR-214 and EZH2 interference on the Wnt/β-catenin signaling pathway (*p < 0.05, **p < 0.01)

Fig. 5

Western blot analysis of the expression levels of β-catenin, Cyclin D1, EZH2, TCF4, and LEF1 and the TA cell differentiation markers Integrin α6, CK15, and CK19 after transfection. The data represent the mean value from three independent experiments

Fig. 6

Immunofluorescence localization of TCF4 and β-catenin following miR-214 transfection. Green color: anti-TCF4; red color: anti-β-catenin; blue color: DAPI staining; yellow: overlap of green and red color. (Color figure online)