Xu Hong, Gu Li-Na, Yang Qian-Yuan, Zhao De-Yu, Liu Feng
Department of Respiratory Medicine, Nanjing Children's Hospital Affiliated to Nanjing Medical University, Nanjing, China.
J Bioenerg Biomembr. 2016 Jun;48(3):293-9. doi: 10.1007/s10863-016-9659-7. Epub 2016 Apr 25.
Mast cells play a pivotal role in the immediate reaction in asthma. In a previous study, it was found that MicroRNA-221 (miR-221) was associated with asthma. Hence, in the present study, the role and the potential mechanisms of miR-221 on immunoglobulin E (IgE)-mediated activation of mast cells degranulation were investigated. MiR-221 expression was first quantified by qRT-PCR in IgE-mediated activation of mast cells. RBL-2H3 cells were then transfected with miR-221 mimic or miR-221 inhibitor, the IgE-mediated degranulation was detected in mast cells. The influence of miR-221 on expression of phospholipase C gamma (PLCγ1), p-PLCγ1, protein kinase B (Akt), phospho-Akt (p-Akt), inhibitor of kappa B (IκB-α), and phospho-IκB-α (p-IκB-α) were examined by Western blot, whereas free calcium ion (Ca(2+)) level was measured by flow cytometry and NF-κB expression was determined by EMSA. Phosphoinositide 3-kinase (PI3K)-inhibitor (LY294002) and NF-κB-inhibitor [pyrrolidine dithiocarbamate (PDTC)] were used to investigate the role of PI3K/Akt pathway and NF-κB in miR-221 promoting IgE-mediated activation of mast cells degranulation. The expression of miR-221 was upregulated in IgE-mediated activation of mast cells, and it was overexpressed in miR-221 mimic transfected cells. The degranulation was found to be significantly increased in miR-221 overexpressed cells while it was found to be significantly decreased in miR-221 downregulated cells. The expression of p-PLCγ1, p-Akt, p-IκB-α as well as NF-κB and Ca(2+) release were increased in miR-221 overexpressed cells. PI3K-inhibitor (LY294002) could rescue the promotion of degranulation caused by miR-221 in IgE-mediated activation of mast cells. However, NF-κB-inhibitor (PDTC) could not rescue the promotion of degranulation caused by miR-221 in IgE-mediated activation of mast cells. MiR-221 promotes IgE-mediated activation of mast cells degranulation by PI3K/Akt/PLCγ/Ca(2+) signaling pathway, in a non-NF-κB dependent manner.
肥大细胞在哮喘的速发反应中起关键作用。在先前的一项研究中，发现微小RNA - 221（miR - 221）与哮喘有关。因此，在本研究中，研究了miR - 221对免疫球蛋白E（IgE）介导的肥大细胞脱颗粒激活的作用及其潜在机制。首先通过qRT - PCR对IgE介导的肥大细胞激活中miR - 221的表达进行定量。然后用miR - 221模拟物或miR - 221抑制剂转染RBL - 2H3细胞，检测肥大细胞中IgE介导的脱颗粒情况。通过蛋白质免疫印迹法检测miR - 221对磷脂酶Cγ（PLCγ1）、磷酸化PLCγ1（p - PLCγ1）、蛋白激酶B（Akt）、磷酸化Akt（p - Akt）、κB抑制蛋白（IκB - α）和磷酸化IκB - α（p - IκB - α）表达的影响，而通过流式细胞术测量游离钙离子（Ca(2+)）水平，并通过电泳迁移率变动分析（EMSA）测定NF - κB表达。使用磷脂酰肌醇3 -激酶（PI3K）抑制剂（LY294002）和NF - κB抑制剂[吡咯烷二硫代氨基甲酸盐（PDTC）]来研究PI3K/Akt途径和NF - κB在miR - 221促进IgE介导的肥大细胞脱颗粒激活中的作用。在IgE介导的肥大细胞激活中miR - 221的表达上调，并且在转染了miR - 221模拟物的细胞中过表达。发现miR - 221过表达的细胞中脱颗粒显著增加，而在miR - 221下调的细胞中脱颗粒显著减少。在miR - 221过表达的细胞中，p - PLCγ1、p - Akt、p - IκB - α以及NF - κB的表达和Ca(2+)释放增加。PI3K抑制剂（LY294002）可以挽救miR - 221在IgE介导的肥大细胞激活中引起的脱颗粒促进作用。然而，NF - κB抑制剂（PDTC）不能挽救miR - 221在IgE介导的肥大细胞激活中引起的脱颗粒促进作用。miR - 221通过PI3K/Akt/PLCγ/Ca(2+)信号通路以非NF - κB依赖的方式促进IgE介导的肥大细胞脱颗粒。
