MicroRNA-214-3p facilitates M2 macrophage polarization by targeting GSK3B

Abstract

Allergic rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa. M2 macrophage polarization can reduce inflammation and repair tissue injury during AR development. Studies have substantiated the involvement of miRNAs in AR pathogenesis. Herein, the molecular mechanism of miR-214-3p in AR development was explored. To mimic the AR environment, ovalbumin (OVA) was used to treat macrophages. MiR-214-3p and glycogen synthase kinase 3 beta (GSK3B) expression in nasal mucus tissues and macrophages was assessed by RT-qPCR. The M2 phenotypic signature of CD206 in macrophages was assessed by flow cytometry. The protein expression of GSK3B and M2 macrophage markers (ARG-1 and IL-10) was evaluated by western blotting. The correlation between miR-214-3p and GSK3B was validated by a luciferase reporter assay. We found that miR-214-3p was overexpressed in macrophages and nasal mucus tissues from AR patients. MiR-214-3p facilitated M2 polarization of macrophages upon OVA stimulation. Mechanistically, miR-214-3p targeted the GSK3B 3' untranslated region in macrophages. In addition, GSK3B was downregulated in macrophages and nasal mucus tissues from AR patients. In rescue assays, GSK3B downregulation reversed the inhibitory effects of miR-214-3p silencing on M2 polarization of macrophages treated with OVA. Overall, miR-214-3p facilitates M2 macrophage polarization by targeting GSK3B.

Keywords: AR; GSK3B; M2 macrophage polarization; MiR-214-3p.

FIGURE 1

MiR‐214‐3p is overexpressed in nasal mucus tissues and macrophages from AR patients. (A) MiR‐214‐3p levels in nasal mucus tissues from healthy controls and AR patients were assessed via RT‐qPCR. (B) FACS was used to sort macrophages derived from the nasal mucus of healthy controls or AR patients. (C) MiR‐214‐3p expression in macrophages of healthy controls and AR patients was assessed by RT‐qPCR. (D) OVA (5 mg/ml) was used to stimulate the sorted macrophages from healthy controls for 6, 12, 24, or 48 h. *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 2

MiR‐214‐3p facilitates OVA‐induced M2 macrophage polarization. (A) Transfection efficiency of miR‐214‐3p inhibitor into macrophages from nasal mucus of healthy controls was assessed by RT‐qPCR. (B,C) proportion of CD206+ macrophages from nasal mucus of healthy controls in four groups (con, OVA, OVA+NC inhibitor, and OVA+miR‐214‐3p inhibitor) by flow cytometry. (D–F) Western blotting was conducted to evaluate M2 macrophage markers (IL‐4, IL‐5, IL‐13, IL‐10, and ARG‐1) protein expression in macrophages from nasal mucus of healthy controls in four groups (con, OVA, OVA+NC inhibitor, and OVA+miR‐214‐3p inhibitor). **p < 0.01, ***p < 0.001

FIGURE 3

MiR‐214‐3p targets GSK3B. (A) Potential target genes (LSM12, RC3H1, NAA15, AMMECR1L, ZFAND3, NEO1, DOLPP1, GSK3B, and MED19) of miR‐214‐3p were predicted by miRDB. (B) The expression of potential downstream mRNAs of miR‐214‐3p in macrophages of nasal mucus after downregulating miR‐214‐3p was assessed by RT‐qPCR. (C) A binding site between miR‐214‐3p and GSK3B was predicted using TargetScan. (D) The interaction between miR‐214‐3p and the GSK3B 3’UTR was validated by a luciferase reporter assay. (E) Western blotting was performed to assess GSK3B protein expression in macrophages from nasal mucus after miR‐214‐3p knockdown. (F) The miR‐214‐3p mimic‐mediated overexpression efficiency of miR‐214‐3p was assessed by RT‐qPCR. (G,H) GSK3B expression in miR‐214‐3p mimic‐transfected macrophages was assessed by RT‐qPCR and western blotting. (I) A luciferase reporter assay was performed to reveal the luciferase activity of GSK3B 3′UTR‐Wt/Mut reporter vectors under the condition of miR‐214‐3p overexpression. **p < 0.01, ***p < 0.001

FIGURE 4

GSK3B is downregulated in nasal mucus tissues and macrophages from AR patients. (A) GSK3B expression in nasal mucus tissues from healthy controls and AR patients was assessed by RT‐qPCR. (B,C) The mRNA and protein levels of GSK3B in macrophages derived from the nasal mucus of AR patients or healthy controls were assessed by RT‐qPCR and western blotting. (D–F) After OVA (5 mg/ml) treatment for 6, 12, 24, or 48 h, the mRNA and protein levels of GSK3B in macrophages from the nasal mucus of healthy controls were examined by RT‐qPCR and western blotting. (G) The correlation between miR‐214‐3p and GSK3B in nasal mucus tissues from AR patients (n = 28) was analyzed by Pearson correlation analysis. *p < 0.05, **p < 0.01

FIGURE 5

GSK3B downregulation reverses the effects of miR‐214‐3p knockdown on M2 macrophage polarization. (A) GSK3B protein expression in OVA‐treated macrophages after silencing GSK3B was assessed by western blotting. (B,C) M2 phenotypic signature marker CD206 in macrophages upon OVA stimulation after silencing miR‐214‐3p or GSK3B was assessed by flow cytometry. (D,E) the protein expression of M2 macrophage markers (IL‐4, IL‐5, IL‐13, IL‐10, and ARG‐1) in OVA‐stimulated macrophages after inhibiting GSK3B or miR‐214‐3p was examined by western blotting. **p < 0.01, ***p < 0.001