MiRNA-221-5p suppressed the Th17/Treg ratio in asthma via RORγt/Foxp3 by targeting SOCS1

Abstract

Background: This study was designed to investigate the mechanism and effects of miRNA-221-5p on the T-helper 17 (Th17)/T-regulatory (Treg) ratio in asthma.

Methods: BALB/c mice were intranasally challenged with 100 µg OVA on 14 and 21 day. Mice were rechallenged with 2.5% OVA-PBS on 22 and 28 day. Mice were sacrificed using on day 30 under 35 mg/kg pentobarbital sodium. PBMCs were induced vitro model of asthma using 500 ng of lipopolysaccharides (LPS) for 4 h.

Results: The expression of miRNA-221-5p was reduced in in vivo model, compared sham group. The vitro model of asthma treated with miRNA-221-5p mimic resulted in the reduction of IL-6, IL-17, IL-21 and IL-22 levels, and induction of IL-10, IL-35 and TGF-β levels. In addition, down-regulation of miRNA-221-5p induced the protein expression of suppressor of cytokine signaling 1 (SOCS1) and receptor-related orphan receptor-gamma-t (RORγt) and suppressed that of FOXP3 in in vitro model of asthma. Over-expression of miRNA-221-5p induced the protein expression of FOXP3, and suppressed that of SOCS1 and RORγt in in vitro model of asthma. The inhibition of SOCS1 or RORγt attenuated the effects of anti-miRNA-221-5p on Th17/Treg ratio in asthma.

Conclusion: miRNA-221-5p may play critical roles in driving the differentiation of Th17/Treg ratio via RORγt/Foxp3 by Targeting SOCS1, reduced the function of Th17 cells by directly inhibiting RORγt/SOCS1 and promoted the function of Treg cells via Foxp3/ SOCS1 in asthma.

Keywords: Asthma; RORγt; SOCS1; Th17/Treg; miRNA-221-5p.

Fig. 1

The expression of miRNA-221-5p in asthma. Th17 cell (A and B), Treg cells (C and D), HE staining (E), inflammation score (F), BALF levels (G), the expression of miRNA-221-5p in asthma by gene chip (H) and PCR (I). Sham, sham control group; Asthma, asthma model group. ^**p < 0.01 versus sham control group

Fig. 2

Over-expression of miRNA-221-5p regulates imbalance of Th17 and Treg Cells in model asthma. The expression of miRNA-221-5p (A), IL-6 (B), IL-17 (C), IL-21 (D), IL-22 (E), IL-10 (F), IL-35 (G) and TGF-β (H) levels. Negative, negative mimics group; miR-221-5p, Over-expression of miRNA-221-5p group. ^**p < 0.01 versus negative mimics group

Fig. 3

Over-expression of miRNA-221-5p regulates imbalance of Th17 and Treg Cells in model asthma through RORγt/Foxp3 by Targeting SOCS1. Heat map (A), network signal (B), Network signal analysis (C), 3’UTR region of SOCS1 showed potential alignment with miRNA-221-5p sequence (D), luciferase reporter activity levels (E). Negative, negative mimics group; miR-221-5p, Over-expression of miRNA-221-5p group. ^**p < 0.01 versus negative mimics group

Fig. 4

Over-expression of miRNA-221-5p regulates SOCS1, RORγt and Foxp3 expression. SOCS1, RORγt and Foxp3 protein expression by statistical analysis (A–C) and western blotting analysis (D). SOCS1 expression using Immunofluorescence (E). Negative, negative mimics group; miR-221-5p, Over-expression of miRNA-221-5p group. ^**p < 0.01 versus negative mimics group

Fig. 5

Down-regulation of miRNA-221-5p regulates imbalance of Th17 and Treg Cells in model asthma through RORγt/Foxp3 by Targeting SOCS1. The expression of miRNA-221-5p (A), IL-6 (B), IL-17 (C), IL-21 (D), IL-22 (E), IL-10 (F), IL-35 (G) and TGF-β (H) levels, SOCS1, RORγt and Foxp3 protein expression by statistical analysis (I–K) and western blotting analysis (L). Negative, negative mimics group; anti-221-5p, down-regulation of miRNA-221-5p group. ^**p < 0.01 versus negative mimics group

Fig. 6

The inhibition of SOCS1 reduced the effects of anti-miRNA-221-5p on Th17/Treg Ratio in asthma. SOCS1, RORγt and Foxp3 protein expression by statistical analysis (A–C) and western blotting analysis (D), IL-6 (E), IL-17 (F), IL-21 (G), IL-22 (H), IL-10 (I), IL-35 (J) and TGF-β (K) levels. Negative, negative mimics group; anti-221-5p, down-regulation of miRNA-221-5p group; si-SOCS1, si-SOCS1 and down-regulation of miRNA-221-5p group. ^**p < 0.01 versus negative mimics group, ^##p < 0.01 versus down-regulation of miRNA-221-5p group

Fig. 7

The inhibition of RORγt reduced the effects of anti-miRNA-221-5p on Th17/Treg Ratio in asthma. RORγt and Foxp3 protein expression by statistical analysis (A and B) and western blotting analysis (C), IL-6 (D), IL-17 (E), IL-21 (F), IL-22 (G), IL-10 (H), IL-35 (I) and TGF-β (J) levels. Negative, negative mimics group; anti-221-5p, down-regulation of miRNA-221-5p group; si-RORγt, si-RORγt and down-regulation of miRNA-221-5p group. ^**p < 0.01 versus negative mimics group, ^##p < 0.01 versus down-regulation of miRNA-221-5p group.

Fig. 8

The inhibition of FOXP3 reduced the effects of miRNA-221-5p on Th17/Treg Ratio in asthma. RORγt and Foxp3 protein expression by statistical analysis (A and B) and western blotting analysis (C), IL-6 (D), IL-17 (E), IL-21 (F), IL-22 (G), IL-10 (H), IL-35 (I) and TGF-β (J) levels. Negative, negative mimics group; anti-221-5p, down-regulation of miRNA-221-5p group; si-FOXP3, si-FOXP3 and down-regulation of miRNA-221-5p group. ^**p < 0.01 versus negative mimics group, ^##p < 0.01 versus down-regulation of miRNA-221-5p group

Fig. 9

MiRNA- Regulates the Th17/Treg Ratio in asthma via RORγt/Foxp3 by Targeting SOCS1