The Effect Of MiRNA-221-3p On The Degranulation Function Of Mast Cells And Its Mechanism

ObjectiveTo explore the effect and mechanisms of miRNA-221-3p（abbreviated as miR-221 below）on mast cell degranulation.MethodsThe rat mast cell lines RBL-2H3 were sensitized with anti-DNP-IgE and stimulated with DNP-HAS（IgE-mediated）to establish mast cell degranulation.The expression of miR-221 was quantified by quantitative real-time polymerase chain reaction（RT-PCR）in IgE-mediated mast cells.RBL-2H3 cells were transfected with miR-221 mimics and the transfection efficiency was verified by RT-PCR.After overexpressing miR-221,the IgE-mediated mast cell degranulation was detected in RBL-2H3 cells.Then the effect of miR-221 overexpression on molecule expression associated with signaling pathway was determined by microarray analysis in IgE-mediated mast cells.The influence of miR-221 overexpression on phosphorylation（p）-PLCγ1 expression was examined by western blot in IgE-mediated mast cells and the calciumion was tested by flow cytometry.The effect of miR-221 overexpression on p-Akt and its downstream p-IκB-α expression were analysed by western blot in IgE-mediated mast cells and NF-κB expression was determined by EMSA.PI3K-inhibitor（LY294002）was used to investigate the role of PI3K/Akt pathway in miR-221 promoting IgE mediated mast cells degranulation.NF-κB-inhibitor（PDTC）was used to investigate the role of NF-κB in miR-221 promoting IgE mediated mast cells degranulation.ResultsThe expression of miR-221 increased in IgE mediated mast cells.miR-221 expression increased in cells transfected with miR-221 mimics and the transfection was successful.Overexpression of miR-221 promoted IgE-mediated mast cell degranulation.Microarray analysis showed that miR-221 overexpression promoted the molecule expression associated with PLCγ and PI3K/AKT signaling pathway in IgE mediated mast cells.In PLCγ signaling pathway,western blot displayed miR-221 overexpression promoted p-PLCγ1 expression in IgE mediated mast cells.Flow cytometry showed miR-221 overexpression promoted the release of calciumions.In PI3K/AKT signaling pathway,western blot revealed miR-221 overexpression promoted p-Akt and p-IκB-α expression in IgE mediated mast cells.Electrophoretic Mobility Shift Assay（EMSA）displayed miR-221 overexpression promoted NF-κB expression.PI3K-inhibitor（LY294002）could rescue the promotion of degranulation caused by miR-221 in IgE mediated mast cells.NF-κB-inhibitor（PDTC）could not rescue the promotion of degranulation caused by miR-221 in IgE mediated mast cells.ConclusionmiR-221 promotes IgE-mediated mast cell degranulation by PI3K/Akt/PLCγ/ Ca²⁺ signaling pathway,non-NF-κB dependent.