MiR-221 promotes IgE-mediated activation of mast cells degranulation by PI3K/Akt/PLCγ/Ca(2+) pathway.
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.
Department of Respiratory Medicine, Nanjing Children's Hospital Affiliated to Nanjing Medical University, Nanjing, China.
Hong
H
Xu
Department of Respiratory Medicine, Nanjing Children's Hospital Affiliated to Nanjing Medical University, Nanjing, China.
Li-Na
LN
Gu
Department of Respiratory Medicine, Nanjing Children's Hospital Affiliated to Nanjing Medical University, Nanjing, China.
Qian-Yuan
QY
Yang
Department of Respiratory Medicine, Nanjing Children's Hospital Affiliated to Nanjing Medical University, Nanjing, China. zhaodeyu988@126.com.
De-Yu
DY
Zhao
Department of Respiratory Medicine, Nanjing Children's Hospital Affiliated to Nanjing Medical University, Nanjing, China. axsliu@163.com.
Feng
F
Liu
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015550:Cell Degranulation / Q000187:drug effects*; D002460:Cell Line; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008407:Mast Cells; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051966:Phospholipase C gamma / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
C576526:MIRN221 microRNA, rat; D035683:MicroRNAs; D016328:NF-kappa B; D007073:Immunoglobulin E; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D051966:Phospholipase C gamma; D002118:Calcium
Degranulation; Mast cell; MiR-221; PI3K/Akt pathway; PLCγ/Ca2+ pathway