miRNA-34b/c regulates mucus secretion in RSV-infected airway epithelial cells by targeting FGFR1

Abstract

Respiratory syncytial virus (RSV) infection in airway epithelial cells is the main cause of bronchiolitis in children. Excessive mucus secretion is one of the primary symbols in RSV related lower respiratory tract infections (RSV-related LRTI). However, the pathological processes of mucus hypersecretion in RSV-infected airway epithelial cells remains unclear. The current study explores the involvement of miR-34b/miR-34c in mucus hypersecretion in RSV-infected airway epithelial cells by targeting FGFR1. First, miR-34b/miR-34c and FGFR1 mRNA were quantified by qPCR in throat swab samples and cell lines, respectively. Then, the luciferase reporters' assay was designed to verify the direct binding between FGFR1 and miR-34b/miR-34c. Finally, the involvement of AP-1 signalling was assessed by western blot. This study identified that miR-34b/miR-34c was involved in c-Jun-regulated MUC5AC production by targeting FGFR1 in RSV-infected airway epithelial cells. These results provide some useful insights into the molecular mechanisms of mucus hypersecretion which may also bring new potential strategies to improve mucus hypersecretion in RSV disease.

Keywords: FGFR1; airway epithelial cells; miRNA; mucus secretion; respiratory syncytial virus.

FIGURE 1

The protein level of FGFR1 in airway epithelia increase significantly after RSV infection. (A) qPCR was performed to examine FGFR1 mRNA level in RSV‐infected HBECs. (B) Western Blot was performed to examine FGFR1 protein level in RSV‐infected HBECs. (C) mRNA expression of FGFR1 in throat swab samples was analysed from RSV patients (RSV, n = 11) and healthy controls (Control, n = 11). **p < 0.05

FIGURE 2

Fibroblast growth factor receptor 1 expression in RSV‐infected patients and healthy controls in public RNA profiling datasets. (A) FGFR1 expression profile graph in GSE105450 and differential expression analysis between RSV patients and healthy controls. (B) FGFR1 expression profile graph in GSE97742 and differential expression analysis between acute RSV patients and discharge RSV patients. (C) FGFR1 expression profile graph in GSE117827 and differential expression analysis between RSV infection patients and asymptomatic controls

FIGURE 3

miR‐34b/miR‐34c overexpression inhibited MUC5AC expression in RSV‐infected airway epithelial cells. (A) Expression of miR‐34b and miR‐34c in RSV‐infected HBECs (B) Expression of miR‐34b and miR‐34c in throat swab samples from RSV patients (n = 11) and healthy controls (n = 11). (C) HBECs was transfected with miR‐34b or miR‐34c mimic, qPCR validate the expression of miR‐34b/miR‐34c after transfection. (D) HBECs treated with miR‐34b mimic and miR‐34c mimics (miR mimic) or negative control mimic (Negative control) were subject to CCK8 assay. (E) The mRNA expression of MUC5AC was analysed by qPCR after the transfection of miR‐34b/miR‐34c mimics in RSV‐infected (RSV+) or control (RSV‐) HBECs. **p < 0.05, ***p < 0.01

FIGURE 4

Fibroblast growth factor receptor 1 is identified as a direct target of miR‐34b/miR‐34c in airway epithelial cells. (A) The predicted miR‐34b/miR‐34c complementary sequence with the 3′‐UTR region of FGFR1, as well as the mutant containing altered nucleotides in the 3′‐UTR of FGFR1. (B) Luciferase activity assay was operated after transfection with FGFR1‐WT or FGFR1‐MUT reporter and miR‐NC or miR‐34b in HBECs. (C) Luciferase activity assay was operated after transfection with FGFR1‐WT or FGFR1‐MUT reporter and miR‐NC or miR‐34c in HBECs. (D) Luciferase activity assay was operated after transfected cells with FGFR1‐WT or FGFR1‐MUT reporter and miR‐NC or miR‐34b in A549 cells. (E) Luciferase activity assay was operated after transfected with FGFR1‐WT or FGFR1‐MUT reporter and miR‐NC or miR‐34c in A549 cells. (F) qPCR was used to analyse the mRNA expression of FGFR1 in RSV‐infected HBECs in the presence of miR‐34b/miR‐34c overexpression. (G) Western blotting was performed to examine the effect of miR‐34b/ miR‐34c overexpression on the protein level of FGFR1 in RSV‐infected HBECs. ****p < 0.001

FIGURE 5

Respiratory syncytial virus mediated activation of c‐Jun/MUC5AC signalling was blocked by FGFR1 inhibition. (A) Western blotting was used to assess the effects of miR‐34b/miR‐34c mimics or FGFR1 inhibitor (PD1730741) on the protein levels of p‐c‐Jun and c‐Jun in RSV‐infected HBECs. (B) qPCR was used to assess the effects of miR‐34b/miR‐34c mimics or PD1730741 on the mRNA levels of MUC5AC in RSV‐infected HBECs. ****p < 0.001