The Influenza A Virus H3N2 Triggers the Hypersusceptibility of Airway Inflammatory Response via Activating the lncRNA TUG1/miR-145-5p/NF-κB Pathway in COPD

Abstract

Background: Patients with chronic obstructive pulmonary disease (COPD) are more susceptible to influenza A virus (IAV) with more severe symptoms, yet the underlying molecular mechanisms of the hypersusceptibility of airway inflammatory response remain unclear. Methods: The primary human bronchial epithelial cells (pHBECs) were isolated from normal and COPD bronchial tissues (NHBE and DHBE) and cultured with/without IAV infection in vitro. DHBE cells were exposed to IAV for 24 h after knockdown of lncRNA TUG1 with short hairpin RNA (shRNA). Gain-of-function assays were performed with the miR-145-5p inhibitor and NF-κBp65 transfection. The expressions of lncRNA TUG1, miR-145-5p, phospho-NF-κBp65, NF-κBp65, TNF-α, and (Interleukin) IL-1β were examined with qRT-PCR, Western blotting, and ELISA. The interactions of lncRNA TUG1, miR-145-5p, and NF-κB were verified with luciferase reporter assay. Results: The expressions of lncRNA TUG1, phospho-NF-κBp65, TNF-α, and IL-1β were increased significantly in pHBECs after being infected with IAV for 24 h (all p0.05). The detailed time analysis revealed that the NF-κBp65 in DHBE was activated earlier than that in NHBE by Western blotting and immunofluorescence. Knockdown of lncRNA TUG1 and miR-145-5p mimic attenuated the expressions of NF-κBp65, TNF-α, and IL-1β significantly. The miR-145-5p inhibitor and NF-κBp65 transfection reversed the attenuated expressions of NF-κBp65, TNF-α, and IL-1β. Conclusion: The IAV causes the hypersusceptibility of airway inflammatory response, which may be closely associated with more severe symptoms in AECOPD patients. The lncRNA TUG1 inhibitor may be a promising therapeutic strategy for AECOPD caused by IAV.

Keywords: COPD; NF-κB; airway inflammatory hypersusceptibility; influenza A virus; lncRNA TUG1; miR-145-5p.

FIGURE 1

IAV activated the NF-κB pathway in pHBECs. (A,A1) The protein expression and relative intensity of NF-κBp65 and phospho-p65 in pHBECs after infection with or without IAV for 24 h. (B,B1) The time-dependent protein expression and relative intensity of NF-κBp65 and phospho-p65 in NHBE after infection with IAV. (C,C1) The time-dependent protein expression and relative intensity of NF-κBp65 and phospho-p65 in DHBE after infection with IAV. (D,E) ELISA analyses of IL-1β and TNF-α in pHBECs after infection with IAV (^* p<0.05). Each dataset comprises three independent experiments.

FIGURE 2

IAV activated the NF-κB pathway earlier in DHBE. (A) Cell immunofluorescence of IAV induced NF-κBp65 nuclear translocation in pHBECs measured at different times. The IAV induced NF-κBp65 nuclear translocation was obviously detected at four hpi in NHBE (left) and two hpi in DHBE (right), scale bar = 50 μm. Note: hpi: hours post infection, (B) NF-κB/p65 expression and relative intensity in the nucleus of pHBECs after infection with IAV for different times.

FIGURE 3

Knockdown of lncRNA TUG1 reduced the IAV induced airway inflammation in DHBE. (A,B) The expression of lncRNA TUG1 and miR-145-5p was analyzed in pHBECs with or without IAV infection for 24 h (note: ^* p<0.05 as compared to NHBE, ^## p<0.01 as compared to NHBE, ^& p<0.05 as compared to DHBE). (C,D) The mRNA levels of lncRNA TUG1 and miR-145-5p were analyzed in DHBE pretreated with or without sh-TUG1. (E,F) The protein expressions of NF-κBp65 and phospho-NF-κBp65 were analyzed in DHBE pretreated with or without sh-TUG1 by Western blotting. (G,H) The protein expressions of IL-1β and TNF-α were analyzed in DHBE pretreated with or without sh-TUG1 by ELISA. (^* p<0.05, ^** p<0.01, ^*** p<0.001 as compared to DHBE + sh-NC group, ^## p<0.01, ^### p<0.001 as compared to DHBE + H3N2 group, ^§§ p<0.01, ^§§§ p<0.001 as compared to DHBE group, ^&& p<0.01, ^&&& p<0.001 as compared to DHBE + sh-NC group, ^$ p<0.05, ^$$ p<0.01 as compared to DHBE + sh-NC + H3N2 group). Each dataset comprises three independent experiments.

FIGURE 4

lncRNA TUG1 positively regulated the IAV induced airway inflammation by sponging miR-145-5p in DHBE. (A) The putative miR-145-5p binding sequence of TUG1 mRNA. (B) Luciferase activity was measured between miR-145-5p (wt and mut) and TUG1 (wt and mut). (C) The putative miR-145-5p binding sequence of the 3′UTR of NF-κBp65. (D) Luciferase activity was measured between miR-145-5p (wt and mut) and NF-κBp65 (wt and mut). (E,F) The protein expressions of NF-κBp65 and phospho-NF-κBp65 were analyzed in DHBE pretreated with or without miR-145-5p mimic by Western blotting. (G,H) The protein expressions of proinflammatory cytokines IL-1β and TNF-α were analyzed in DHBE pretreated with or without miR-145-5p mimic by ELISA (^* p<0.05, ^** p<0.01 as compared to DHBE + miR-NC group, ^## p<0.01, ^### p<0.001 as compared to DHBE + H3N2 group,^§§ p<0.01, ^§§§ p<0.001 as compared to DHBE group, ^&& p<0.01, ^&&& p<0.001 as compared to DHBE+ miR-NC group, ^$$ p<0.01 as compared to DHBE+ miR-NC +H3N2 group). Each dataset comprises three independent experiments. (I,J) The protein expressions of NF-κBp65 and phospho-NF-κBp65 were analyzed in DHBE pretreated with or without sh-TUG1/miR-145-5p inhibitor by Western blotting. (K,L) The protein expressions of proinflammatory cytokines IL-1β and TNF-α were analyzed in DHBE pretreated with or without sh-TUG1/miR-145-5p inhibitor by ELISA (^** p<0.01, ^*** p<0.001 as compared to DHBE group, ^## p<0.01, ^### p<0.001 as compared to DHBE + sh-NC + H3N2 group, ^$ p<0.05, ^$$ p<0.01 as compared to DHBE+ sh-TUG1+H3N2 group, ^§ p<0.05, ^§§ p<0.01, ^§§§ p<0.001 as compared to DHBE+ sh-TUG1+miR-145-5p in+H3N2 group). Each dataset comprises three independent experiments.

FIGURE 5

Overexpression of NF-κBp65 reversed the effects of lncRNA TUG1 knockdown in DHBE after infection with IAV. (A,B) The protein expressions of NF-κBp65 and phospho-NF-κBp65 were analyzed in DHBE pretreated with or without sh-TUG1 and NF-κBp65 plasmid by Western blotting. (C,D) The protein expressions of proinflammatory cytokines IL-1β and TNF-α were analyzed in DHBE pretreated with or without sh-TUG1 and NF-κBp65 plasmid by ELISA (^** p<0.01, ^*** p<0.001 as compared to DHBE group, ^## p<0.01, ^### p<0.001 as compared to DHBE + sh-NC + H3N2 group, ^$$ p<0.01 as compared to DHBE + sh-TUG1+H3N2 group, ^§ p<0.05, ^§§ p<0.01, ^§§§ p<0.001 as compared to DHBE + sh-TUG1+p65 + H3N2 group). Each dataset comprises three independent experiments.

FIGURE 6

Schematic diagram to illustrate the interactions of lncRNA TUG1/miR-145-5p/NF-κB pathway.