Study On Regulatory Effect Of MicroRNA-146A On The MUC5AC In Chronic Rhinitis-sinusitis

Objective:We investigated microRNA-146a(miR-146a),Human neutrophil elastase(HNE),Epidermal growth factor receptor(EGFR),Mucins 5AC(MUC5AC)expression in the nasal mucosa of CRS patients,also observed miR-146 a and MUC5 AC expression in vitro cell culture experiments.This study was designed to explore the effects and role of miR-146 a on the MUC5 AC production stimulated by HNE in CRS patient.Methods:1?Fluorescence quantitative PCR(q-PCR)experiment was used to observe the expression of miR-146 a,HNE,EGFR,MUC5 AC level in CRS group(30 patients)and control group(15 patients).2?The cultured human nasal epithelial cells were exposed to different concentration and different treatment times of HNE,q-PCR experiment and Enzyme linked immunosorbent assay(ELISA)was used to evaluated the expression of MUC5 AC protein level in the supernatant fluid,the expression of p-EGFR protein and EGFR protein were detected by western blot(WB).3?The cultured human nasal epithelial cells were exposed to EGFR sepecific inhibitor(AG1478).And then we evaluated the expression of EGFR mRNA and MUC5 AC protein level in the supernatant fluid by using q-PCR experiment and ELISA method respectively.4?The cultured human nasal epithelial cells were transfected with miR-146 a mimics or Negative Control(NC).We evaluated the expression of miR-146 a and MUC5 AC mRNA by using q-PCR experiment,detected MUC5 AC protein level in the supernatant fluid and the ratio of p-EGFR protein to EGFR protein by using ELISA and WB method respectively.5?The 3'UTR of EGFR was cloned into the pGL3-control,then the pGL3-EGFR-3'UTR luciferase reporter plasmid was successfully constructed.The recombinant plasmid vector and miR-146 a mimics were transfected into human nasalepithelial cells,the luciferase activities were detected by Dual-Luciferase Reportor System.Results:1?When compared to the control group,the q-PCR experiment shows that in all patients with CRS,miR-146 a was specifically down-regulated,while HNE,EGFR,MUC5 AC mRNA were up-regulated.Furthermore,the miR-146 a,HNE mRNA,EGFR mRNA,MUC5 AC mRNA expression were not found to have significant changes between the CRS with or without nasal polyps samples.2?In vitro cultured human nasal epithelial cells,the expression of the MUC5 AC level were up-regulated in the exposed to HNE(10nM,24h)group,it detected by q-PCR and ELISA experiment.Furthermore,in the exposed to HNE(24h)group,the ratio of p-EGFR protein to EGFR protein was upper than the control group,it detected by WB experiment.3?In human nasal epithelial cells,we found that in the EGFR sepecific inhibitor(AG1478)group,EGFR mRNA was down-regulated detected by the q-PCR experiment,MUC5 AC protein level in the supernatant fluid were down-regulated detected by ELISA method.4?Transfection human nasal epithelial cells with miR-146 a mimics of 24 h,and then the cells were exposed to HNE,compared to the control,results showed that,in miR-146 a mimics transfected group,the miR-146 a was specifically up-regulated,while MUC5 AC mRNA was specifically down-regulated,it detected by q-PCR experiment.Moreover,the ELISA experiment shows that the expression of MUC5 AC protein level in the supernatant fluid were down-regulated.Furthermore,in miR-146 a mimics transfected group,the ratio of p-EGFR protein to EGFR protein was lower than the control group,it detected by WB method.5?The results of Dual-Luciferase Reportor System showed that the luciferase activities were apparently inhibited in the miR-146 a mimics+ pGL3-EGFR-3'UTR group compared with miR-146 a mimics+ pGL3 group,it can proved that EGFR is a target gene for miR-146 a.Conclusion:Taken together,in CRS patients the miR-146 a depletion on MUC5 ACexpression may be mediated through repressed the activation of EGFR,and EGFR is a target gene for miR-146 a.