Effects Of SEC3on Mucin Secretion Induced By Neutrophil Elastase In Airway Epithelium Cells

Objective：To explore the effects of exocyst complex subunit Sec3on secretion ofmucin（MUC）5AC induced by neutrophil elastase(NE) in human airwayepithelial cells.Methods：1、The16HBE airway epithelial cells were cultured and divided into8groups:GroupA: negative control group；GroupB: epidermal growth factor receptor（EGFR） phosphorylatedinhibitor (AG1487) group；GroupC: Sec3siRNA group；GroupD: negative siRNA control group；GroupE: NE stimulation group；GroupF: epidermal growth factor receptor（EGFR） phosphorylatedinhibitor (AG1487) group treated with NE；GroupG: Sec3siRNA group treated with NE； GroupH: negative siRNA control group treated with NE.2、Choose the neutrophil elastase（NE） as stimulating factor toestablish the hypersecretion model. The level of MUC5AC protein insupernatant, MUC5AC mRNA in culture cells and phosphorylatedepidermal growth factor receptor (p-EGFR) were detected withenzyme-linked immunosorbent assay(ELISA), reversetranscription-polymerase chain reaction (RT-PCT) and Western-blotrespectively.3、Using EGFR phosphorylated inhibitor（AG1487） block the EGFRsignal pathway，detect the level of p-EGFR in AG1487group and controlgroup with Western-blot，and analyse the level of expression of MUC5ACin the two groups.4、Transfecting of the Sec3siRNA to inhibit the function of Sec3，detect the level of Sec3in Sec3group and control group withWestern-blot，confirm the transfection succeed，then analyse the level ofexpression and exocytosis of MUC5AC in the two groups.Results：1、The relative contents of MUC5AC protein in supernatan（tgroup E：0.80±0.09），MUC5AC mRNA（group E：0.75±0.05）and pEGFR（groupE：0.87±0.12）in culture cells in the NE-stimulated group, all hadsignificantly risen when compared with normal control group(p﹤0.01).2、After the addition of EGFR phosphorylated inhibitor，the effect ofNE were suppressed，the level of transcription (group F：0.38±0.06) and secretion （group F：0.32±0.08） of MUC5AC had significantly decreasedwhen compared with control group(p﹤0.01).3、The siRNA of Exocyst complex subunit Sec3only inhibited thesecretion of MUC5AC （group G：0.36±0.09）(p﹤0.01), and didn’teffect the transcription process（group G：0.72±0.04）(p﹥0.05).Conclusion：In the process of MUC5AC hypersecretion induced by neutrophilelastase in human airway epithelial cells, EGFR is the main signal pathwayto mediate the synthesis of MUC5AC，and exocyst complex subunit Sec3play a critical role for mucin exocytosis.