Figures & data
Figure 1. MiR-146a-5p is lowly expressed in the blood samples and HSAECs treated with PAFA
(A) Heat map showed the difference of miRNA expression profile between the bronchial epithelium of asthma patients and healthy volunteers in GSE142237. (B) qRT-PCR was adopted to detect the expression of miR-146a-5p in the plasma of healthy volunteers and asthma patients. (C) ROC curve was plotted to evaluate the diagnostic value of plasma miR-146a-5p in asthma. (D) qRT-PCR was adopted to detect the expression of miR-146a-5p in HSAECs with or without PAF stimulation. (E) ELISA was used to detect the levels of IL-1β, IL-6 and TNF-α in the supernatnat of HSAECs treated with PAF. (F) The mRNA expression of IL-1β, IL-6 and TNF-α in HSAECs treated with PAF was detected by qRT-PCR. (G) With the fluorescence yellow transmittance as an index, the permeability of HSAECs was detected by Transwell assay. All experiments were performed in triplicate. **P < 0.01, and ***P < 0.001.
Figure 2. The effect of miR-146a-5p on airway inflammation and epithelial cell barrier injury in asthma
(A) The mRNA expression miR-146a-5p in PAF-treated HSAECs transfected with miR-146a-5p mimics or inhibitors was detected by qRT-PCR. (B) IL-1β, IL-6 and TNF-α levels in the supernatnat of PAF-treated HSAECs transfected with miR-146a-5p mimics or inhibitors were detected by ELISA. (C) The mRNA expressions of IL-1β, IL-6 and TNF-α in PAF-treated HSAECs transfected with miR-146a-5p mimics or inhibitors were detected by qRT-PCR. (D) The permeability of PAF-treated HSAECs transfected with miR-146a-5p mimics or inhibitors was detected by Transwell assay. (E) and (F) Flow cytometry was used to detect the apoptosis of PAF-treated HSAECs transfected with miR-146a-5p mimics or inhibitors. All experiments were performed in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 3. TRAF6 is the downstream target of miR-146a-5p in HSAECs
(A) Bioinformatics analysis predicted the binding sequence between miR-146a-5p and TRAF6 3'-UTR. (B) Dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-146a-5p and TRAF6. (C) and (D) qRT-PCR and western blot were used to detect the expression of TRAF6 mRNA and the protein level in PAF-treated HSAECs transfected with miR-146a-5p mimics or inhibitors. All experiments were performed in triplicate. **P < 0.01, and ***P < 0.001.
Figure 4. MiR-146a-5p regulates the inflammation and injury of HSAECs by targeting TRAF6
(A) and (B) qRT-PCR and western blot were used to detect the transfection efficiency of miR-146a-5p mimics and TRAF6 overexpression plasmids in PAF-treated HSAECs. (C) and (D) After transfection, ELISA and qRT-PCR were used to detect the expression of IL-1β, IL-6 and TNF-α in PAF-treated HSAECs. (E) and (F) The protein levels of p-ERK, p-JNK, ERK and JNK in PAF-treated HSAECs were detected by Western blot. (G) Transwell assay was used to detect the permeability of PAF-treated HSAECs. (H) and (I) The apoptosis rate of PAF-treated HSAECs was analyzed by flow cytometry. All experiments were performed in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001.
