Long non-coding RNA H19 deficiency ameliorates bleomycin-induced pulmonary inflammation and fibrosis

Abstract

Background: The poor understanding of pathogenesis in idiopathic pulmonary fibrosis (IPF) impaired development of effective therapeutic strategies. The aim of the current study is to investigate the roles of long non-coding RNA H19 (lncRNA H19) in the pulmonary inflammation and fibrosis of IPF.

Methods: Bleomycin was used to induce pulmonary inflammation and fibrosis in mice. The mRNAs and proteins expression in lung tissues was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. H19 knockout (H19^-/-) mice were generated by CRISPR/Cas9.

Results: The expression of H19 mRNA was up-regulated in fibrotic lungs patients with IPF as well as in lungs tissues that obtained from bleomycin-treated mice. H19^-/- mice suppressed bleomycin-mediated pulmonary inflammation and inhibited the Il6/Stat3 signaling. H19 deficiency ameliorated bleomycin-induced pulmonary fibrosis and repressed the activation of TGF-β/Smad and S1pr2/Sphk2 in the lungs of bleomycin-treated mice.

Conclusions: Our data suggests that H19 is a profibrotic lncRNA and a potential therapeutic target for IPF.

Keywords: Pulmonary fibrosis; S1pr2; Smad; lncRNA H19.

Fig. 1

H19 increased in lungs of pulmonary fibrosis. a Data for H19 mRNA expression extracted from the GEO database, comparing whole extracts of lung tissue from human patients diagnosed with IPF (n = 50) and other interstitial lung diseases (ILD) (n = 35) and from control subjects (n = 55) (GSE47460). b Data for H19 mRNA expression extracted from the GEO microarray database, comparing whole extracts of lung tissue from bleomycin-induced lung fibrosis in rat (n = 5 animals per bleomycin-treated group and n = 22 for saline controls). c Representative images of FISH targeting H19 (red) and immunofluorescence (IF) staining of Sftpc in lungs of Sham and bleomycin-treated mice. d The relative H19 mRNA levels in the lungs of Sham wild type (Wt, n = 8), Sham H19 knockout (H19^−/−, n = 6) Wt bleomycin (BLM, n = 8) and H19^−/− BLM (n = 10) mice were determined by real-time RT-PCR. The Hprt1 was used as an internal control. e Representative images of the DNA agarose gels of H19 and Hprt1

Fig. 2

H19 knockout inhibited bleomycin-induced pulmonary inflammation in mice. a Representative immunofluorescence (IF) images of CD45 positive cells in lungs of Sham Wt (n = 10), Sham H19^−/− (n = 7), Wt BLM (n = 9) and H19^−/− BLM (n = 9) mice. b The relative mRNA levels of the inflammatory marker genes including F4/80, CD11b, Ccl20, IL1b and Ym1, in the lungs of Sham Wt (n = 10), Sham H19^−/− (n = 7), Wt BLM (n = 9) and H19^−/− BLM (n = 9) mice. The Hprt1 was used as an internal control. c Representative images of the immune blots of Il6, p-Stat3, Stat3 and β-Actin. d Relative protein expression levels of Il6 and p-Stat3 were normalized using β-actin or Stat3. Statistical significance: *p < 0.05; **p < 0.01; ns not significant

Fig. 3

H19 deficiency ameliorated bleomycin-induced pulmonary fibrosis in mice. a Representative images of hematoxylin–eosin (H&E) staining, masson’s trichrome staining, Sirius red staining and collagen I immunofluorescence staining of Sham Wt, Sham H19^−/−, Wt BLM and H19^−/− BLM mice. b Ashcroft score for Sham Wt (n = 10), Sham H19^−/− (n = 7), Wt BLM (n = 9) and H19^−/− BLM (n = 9) mice. c Quantifying collagen content with hydroxyproline assay in the lungs of Sham Wt, Sham H19^−/−, Wt BLM and H19^−/− BLM mice. Each group, n = 6–8. d Real-time PCR analysis for Tgfb1, Col1a1 and Acta2 in the lungs of Sham Wt, Sham H19^−/−, Wt BLM and H19^−/− BLM mice. Each group, n = 8–10. e Western blot analysis for collagen type I the lungs of Sham Wt, Sham H19^−/−, Wt BLM and H19^−/− BLM mice. f Quantification of the panel (e)

Fig. 4

H19 depletion altered the TGF-β/Smads and S1pr2/Sphk2 pathways in lungs of bleomycin-induced mice. a Western blot analysis for Smad1/5, Smad2, Smad3, Samd4 and their phosphorylated forms the lungs of Sham Wt, Sham H19^−/−, Wt BLM and H19^−/− BLM mice. b Quantification of the panel (a). c Western blot analysis for S1pr2 and Sphk2 in the lungs of Sham Wt, Sham H19^−/−, Wt BLM and H19^−/− BLM mice. d Quantification of the panel (c)

Fig. 5

H19 depletion reduced cell proliferation in lungs of bleomycin-induced mice. a Representative images of co-staining for Sftpc and Ki67 in the lungs of Sham Wt, Sham H19^−/−, Wt BLM and H19^−/− BLM mice. b Western blot analysis for Sftpc, p-Egfr, Egfr, Lin28 and β-actin in the lungs of Sham Wt, Sham H19^−/−, Wt BLM and H19^−/− BLM mice