MicroRNA-126 accelerates IgE-mediated mast cell degranulation associated with the PI3K/Akt signaling pathway by promoting Ca2+ influx

Abstract

Mast cells (MCs) have been reported to serve a crucial role in allergic diseases, including asthma, allergic rhinitis and anaphylaxis. A previous study revealed that microRNA-126 (miR-126) was associated with airway hyperresponsiveness induced by house dust mites, however the molecular mechanisms were unclear. The present study aimed to investigate the effect of miR-126 on immunoglobulin E (IgE)-regulated MC degranulation and explore its underlying mechanisms. miR-126 expression was quantified using a rat model in vivo and in rat peritoneal mast cells (RPMCs) in vitro. Overexpression or downregulation of miR-126 was established by transfection with miR-126 mimics or miR-126 inhibitors and MC degranulation was subsequently evaluated. The effect of miR-126 on protein kinase B (Akt) and phosphorylated Akt protein expression was examined by western blot analysis. The phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) was used to determine the role of the PI3K/Akt signaling pathway. In addition, cytosolic calcium (Ca²⁺) levels were measured by a fura-2 assay. The results demonstrated that miR-126 expression was upregulated in the ear tissues of rats with allergic contact dermatitis and IgE-activated MCs. The overexpression of miR-126 in RPMCs was established following miR-126 mimic transfection. The release of β-hexosaminidase and histamine, markers of MC degranulation, were significantly increased in cells with miR-126 overexpression. The phosphorylation of Akt was significantly increased following transfection with miR-126 mimics in stimulated cells, however the signaling activation was abrogated by LY294002. In addition, Ca²⁺ influx was significantly promoted in stimulated RPMCs overexpressing miR-126. These results indicate that miR-126 accelerated IgE-mediated MC degranulation associated with the PI3K/Akt signaling pathway by promoting Ca²⁺ influx. This suggests that miR-126 may be a promising therapeutic target for the treatment of allergic skin diseases.

Keywords: Ca2+ influx; allergic skin diseases; mast cells; microRNA-126; phosphoinositide 3-kinase/protein kinase B signaling pathway.

Figure 1.

miR-126 expression in rats with 2,4-dinitrofluorobenzene-induced ACD and IgE-activated MCs. (A) miR-126 expression in the left ear tissues of ACD rats. (B) Rat peritoneal MCs were sensitized with anti-DNP IgE (0.5 µg/ml) and stimulated with DNP-HSA (100 ng/ml) for 4 h. The expression of miR-126 was analyzed. Data are presented as the mean ± standard deviation (n=4); *P<0.05. miR, microRNA; IgE, immunoglobulin E; ACD, allergic contact dermatitis; MC, mast cell; DNP, dinitrophenyl; HAS, human serum albumin.

Figure 2.

miR-126 is overexpressed in transfected MCs. Rat peritoneal MCs were transfected with miR-126 mimics, the NC and miR-126 inhibitors. Following 24 h, the expression of miR-126 was examined by reverse transcription-quantitative polymerase chain reaction and analyzed using the 2^−ΔΔCq method. Data are presented as the mean ± standard deviation (n=4); ***P<0.001 and ^##P<0.01. miR, microRNA; MC, mast cell; NC, negative control.

Figure 3.

Effect of miR-126 on MC degranulation. Rat peritoneal MCs were transfected with miR-126 mimics and their NC, as well as the miR-126 inhibitor and its NC. Following sensitization with anti-DNP immunoglobulin E (0.5 µg/ml), the cells were treated with the indicated concentrations of DNP-HSA for 4 h. The release of β-hexosaminidase was detected in the (A) miR-126 mimics and (B) miR-126 inhibitor groups. The release of histamine was detected in the (C) miR-126 mimics and (D) miR-126 inhibitor groups. Data are presented as the mean ± standard deviation (n=4); *P<0.05 and **P<0.01. miR, microRNA; MC, mast cell; NC, negative control; INNC, inhibitor negative control; DNP, dinitrophenyl; HAS, human serum albumin.

Figure 4.

miR-126 activates the PI3K/Akt signaling pathway in IgE-mediated MC degranulation. Rat peritoneal MCs were transfected with miR-126 mimics or its NC. Then the cells were sensitized with anti-DNP IgE (0.5 µg/ml) and stimulated with DNP-HSA (100 ng/ml) for 15 min. The protein expression of Akt and p-Akt were determined by western blot analysis and representative results are reported. Data are presented as the mean ± standard deviation (n=4); *P<0.05. miR, microRNA; MC, mast cell; IgE, immunoglobulin E; NC, negative control; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase; p-, phosphorylated; DNP, dinitrophenyl; HAS, human serum albumin.

Figure 5.

Effect of the PI3K inhibitor (LY294002) in miR-126-improving MC degranulation. Rat peritoneal MCs were transfected with miR-126 mimics or its NC, and the cells were sensitized overnight with anti-DNP immunoglobulin E (0.5 µg/ml). The cells were subsequently treated with DNP-HSA (100 ng/ml) for 4 h in the presence or absence of LY294002 (10 µM). Release of (A) β-hexosaminidase and (B) histamine was measured. Data are presented as the mean ± standard deviation (n=4); *P<0.05. miR, microRNA; MC, mast cell; NC, negative control; PI3K, phosphatidylinositol 3-kinase; DNP, dinitrophenyl; HAS, human serum albumin.

Figure 6.

Ca²⁺ influx was associated with the regulation of miR-126 on MC degranulation. Rat peritoneal MCs were transfected with miR-126 mimics or its NC. The cells were subsequently sensitized with anti-DNP immunoglobulin E (0.5 µg/ml) and treated with DNP-HSA (100 ng/ml) for 4 h. (A) The intracellular Ca²⁺ influx was determined and (B) statistically analyzed using the 340/380 ratio. Representative traces of intracellular Ca²⁺ changes were observed. Data are presented as the mean ± standard deviation (n=4); *P<0.05. miR, microRNA; MC, mast cell; NC, negative control; DNP, dinitrophenyl; HAS, human serum albumin.