Influenza A virus infection dysregulates the expression of microRNA-22 and its targets; CD147 and HDAC4, in epithelium of asthmatics

Abstract

Background: Specific microRNAs (miRNAs) play essential roles in airway remodeling in asthma. Infection with influenza A virus (IAV) may also magnify pre-existing airway remodeling leading to asthma exacerbation. However, these events remain to be fully defined. We investigated the expression of miRNAs with diverse functions including proliferation (miR-20a), differentiation (miR-22) or innate/adaptive immune responses (miR-132) in primary bronchial epithelial cells (pBECs) of asthmatics following infection with the H1N1 strain of IAV.

Methods: pBECs from subjects (n = 5) with severe asthma and non-asthmatics were cultured as submerged monolayers or at the air-liquid-interface (ALI) conditions and incubated with IAV H1N1 (MOI 5) for up to 24 h. Isolated miRNAs were subjected to Taqman miRNAs assays. We confirmed miRNA targets using a specific mimic and antagomir. Taqman mRNAs assays and immunoblotting were used to assess expression of target genes and proteins, respectively.

Results: At baseline, these miRNAs were expressed at the same level in pBECs of asthmatics and non-asthmatics. After 24 h of infection, miR-22 expression increased significantly which was associated with the suppression of CD147 mRNA and HDAC4 mRNA and protein expression in pBECs from non-asthmatics, cultured in ALI. In contrast, miR-22 remained unchanged while CD147 expression increased and HDAC4 remained unaffected in cells from asthmatics. IAV H1N1 mediated increases in SP1 and c-Myc transcription factors may underpin the induction of CD147 in asthmatics.

Conclusion: The different profile of miR-22 expression in differentiated epithelial cells from non-asthmatics may indicate a self-defense mechanism against aberrant epithelial responses through suppressing CD147 and HDAC4, which is compromised in epithelial cells of asthmatics.

Keywords: Airway remodeling; Epithelial cells; Influenza A virus; Severe asthma; microRNA.

Fig. 1

Effect of IAV H1N1 infection on miRNA expression in pBECs from non-asthmatics and asthmatics cultured as monolayers. Cells were infected with IAV H1N1 (MOI 5) and the expression of candidate miRNAs were assessed at different time points (0, 1, 4, 6 and 24 h). a miR-20a, (b) miR-132, and (c) miR-22, were expressed at similar levels in non-asthmatics and asthmatic cells, at baseline (0 h) or after IAV H1N1 infection (1–24 h), using Kruskal Wallis multiple comparisons test, N = 5. The cycle threshold (Ct) value was normalized to that of the RNU44 (∆Ct). Data are presented relative to corresponding non-asthmatic levels at baseline

Fig. 2

Effect of IAV H1N1 infection on miRNA expression in pBECs from non-asthmatics and asthmatics cultured at ALI. Cells were infected with IAV H1N1 (MOI 5) and the expression of candidate miRNAs were assessed at different time points (0, 6, 8 and 24 h). a miR-20a, (b) miR-132, and (c) miR-22, were expressed at similar levels in non-asthmatics and asthmatic cells, at baseline (0 h). H1N1 infection had no effect on the expression of (a) miR-20a, and (b) miR-132, whereas (c) miR-22 expression increased after infection in cells form non-asthmatics but not in asthmatics, *P ≤ 0.05, using the Kruskal Wallis multiple comparisons test, N = 5. The cycle threshold (Ct) value was normalized to that of the RNU44 (∆Ct). Data are presented relative to corresponding non-asthmatic levels at baseline

Fig. 3

Effect of ectopic miR-22 mimic on CD147 and HDAC4 in pBECs of non-asthmatics and asthmatics. pBECs cultured as monolayers were transfected with miR-22 mimic (5 nM) for 24 or 48 h. a-b Transfection was confirmed by assessment of miR-22 expression, and the miR-22 mimic increased miR-22 expression after 24 h in both groups. c-f CD147 mRNA expression was reduced after 24 h in asthmatics and protein expression was suppressed after 48 h in both group. g-j HDAC4 mRNA expression was reduced in asthmatics after 24 h and protein expression was suppressed after 48 h in non-asthmatics. *P ≤ 0.05, using Mann-Whitney nonparametric test, N ≥ 3

Fig. 4

CD147 expression in pBECs from non-asthmatics and asthmatics cultured at ALI. Cells were infected with IAV H1N1 (MOI 5) and the expression of CD147 was assessed at different time points. a and (b) represent mRNA expression of CD147 at baseline (0 h) and after IAV H1N1 infection (6–24 h) in non-asthmatics and asthmatics, respectively. The cycle threshold (Ct) value was normalized to 18S gene (∆Ct) and data are presented relative to corresponding non-asthmatics at baseline. *P ≤ 0.05 intra-cohort, and #P ≤ 0.05 inter-cohort at 24 h, using the Kruskal Wallis multiple comparisons test and Mann-Whitney test, respectively, N=5. c and (d) are immunoblots representative of baseline and 24 h after infection in non-asthmatics and asthmatics, respectively. e and (f) represent densitometric quantification of immunoblots. Data are presented relative to corresponding non-asthmatic levels at baseline. *P ≤ 0.05 intra-cohort, and #P ≤ 0.05 inter-cohort at 24 h, using Mann-Whitney test, N=4

Fig. 5

HDAC4 expression in pBECs from non-asthmatics and asthmatics cultured at ALI. Cells were infected with IAV H1N1 (MOI 5) and HDAC4 expression was assessed at different time points. a and (b) represent mRNA expressions of HDAC4 at baseline and after IAV H1N1 infection (6–24 h) in non-asthmatics and asthmatics, respectively. The cycle threshold (Ct) value was normalized to 18S gene (∆Ct) and data are presented relative to corresponding non-asthmatics at baseline. *P ≤ 0.05 intra-cohort, and #P ≤ 0.05 inter-cohort between corresponding time points, using the Kruskal Wallis multiple comparisons test and Mann-Whitney test, respectively, N=5. c and (d) are immunoblots representative of baseline and 24 h after infection in non-asthmatics and asthmatics, respectively. e and (f) represent densitometric quantification of immunoblots. Data are presented relative to corresponding non-asthmatic levels at baseline. *P ≤ 0.05, using Mann-Whitney test, N=4

Fig. 6

MMP-9 expression and activity in pBECs from non-asthmatics and asthmatics cultured at ALI. Cells were infected with IAV H1N1 (MOI 5) and MMP-9 expression was assessed at different time points. a and (b) mRNA expression of MMP-9 at baseline and after IAV H1N1 infection (6–24 h) in non-asthmatics and asthmatics. The cycle threshold (Ct) value was normalised to the 18S gene (∆Ct) and data are presented relative to corresponding non-asthmatics at baseline. *P ≤ 0.05 intra-cohort, and #P ≤ 0.05 inter-cohort between corresponding time points, using the Kruskal Wallis multiple comparisons test and Mann-Whitney test, respectively, N = 5. c and (d) are immunoblots representative of baseline and 24 h after infection in non-asthmatics and asthmatics, respectively. e and (f) Densitometric quantification of MMP-9 immunoblots. Data are presented relative to corresponding non-asthmatic levels at baseline. *P ≤ 0.05, using non-parametric test, N ≥ 4. g and (h) Representative zymograms after infection (0–24 h) in non-asthmatics and asthmatics. i and (j) Densitometric quantification of zymograms. Data are presented relative to corresponding non-asthmatic levels at baseline. *P ≤ 0.05 intra-cohort, and #P ≤ 0.05 inter-cohort between corresponding time points, using the Kruskal Wallis multiple comparisons test, N = 5

Fig. 7

c-Myc expression in pBECs from non-asthmatics and asthmatics cultured at ALI. Cells were infected with IAV H1N1 (MOI 5) and the expression of c-Myc was assessed at different time points. a and (b) represent mRNA expression of c-Myc at baseline and after IAV H1N1 infection (6–24 h) in non-asthmatics and asthmatics, respectively. The cycle threshold (Ct) value was normalized to 18S gene (∆Ct) and data are presented relative to corresponding non-asthmatics at baseline. *P ≤ 0.05 intra-cohort, and #P ≤ 0.05 inter-cohort at 24 h, using the Kruskal Wallis multiple comparisons test and Mann-Whitney test, respectively, N=5. c and (d) are immunoblots representative of baseline and 24 h after infection in non-asthmatics and asthmatics, respectively. e and (f) represent densitometric quantification of immunoblots. Data are presented relative to corresponding non-asthmatic levels at baseline, N = 4

Fig. 8

SP1 expression in pBECs from non-asthmatics and asthmatics cultured at ALI. Cells were infected with IAV H1N1 (MOI 5) and SP1 expression was assessed at different time points. a and (b) represent mRNA expression of SP1 at baseline and after IAV H1N1 infection (6–24 h) in non-asthmatics and asthmatics, respectively. The cycle threshold (Ct) value was normalized to 18S gene (∆Ct) and data are presented relative to corresponding non-asthmatics at baseline. *P ≤ 0.05 intra-cohort, and #P ≤ 0.05 inter-cohort at 24 h, using Kruskal Wallis multiple comparisons test and Mann-Whitney test, respectively, N=5. c and (d) are immunoblots representative of baseline and 24 h after infection in non-asthmatics and asthmatics, respectively. e and (f) represent densitometric quantification of immunoblots. Data are presented relative to corresponding non-asthmatic levels at baseline, N = 4