Inhibition and role of let-7d in idiopathic pulmonary fibrosis

Abstract

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation.

Objectives: To determine changes in expression and role of microRNAs in IPF.

Methods: RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry.

Measurements and main results: Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells.

Conclusions: Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).

Trial registration: ClinicalTrials.gov NCT00258544.

Figures

Figure 1.

MicroRNAs are differentially expressed in idiopathic pulmonary fibrosis (IPF). (A) The heat map on the left represents global microRNA expression. The heat map on the right represents statistically significant (P < 0.05), differentially expressed microRNAs. Up-regulated microRNAs are shown in progressively brighter shades of yellow, depending on the fold difference, and down-regulated microRNAs are shown in progressively brighter shades of purple. Gray, expression of microRNAs that show no difference between the two groups being compared. The names of the down-regulated microRNAs are provided to the right of the heat map. (B) Quantitative real-time polymerase chain reaction (qRT-PCR) confirmation of the microarray results demonstrates significant changes in all qRT-PCR–measured microRNAs (P < 0.05).

Figure 2.

let-7d is a transforming growth factor (TGF)-β target molecule. (A) Putative SMAD3 (mothers against decapentaplegic homolog-3)-binding sites identified by the Footer algorithm upstream of hsa-let-7d, and identified to be differentially expressed in idiopathic pulmonary fibrosis (IPF) versus control lung. HS = human sequence; MM = mouse sequence. (B) A549 cells were treated with recombinant TGF-β (3 ng/ml) and let-7d expression was determined 0, 2, and 6 hours after stimulation. Results represent the average expression and SD of triplicate experiments. (C) Electrophoretic mobility shift assay and (D) supershift assay of recombinant SMAD3 protein and nuclear extracts isolated from A549 cells treated with recombinant human TGF-β (2 ng/ml) for 1 hour. Comp. DNA = competitor DNA; pAB = polyclonal antibody. Arrow in C, gelshift band representing the protein-DNA complex; Arrow in D, supershift band representing the DNA-protein-antibody complex. (C) and (D) are from two gels run separately. (E) SMAD3 chromatin immunoprecipitation assay revealed association with let-7d in A549 lung cells. IP = immunoprecipitation. (F) Reporter assays were performed on A549 cells transfected with a recombinant vector containing the base pair (bp) −1600 to +87 let-7d region 5′ to the luciferase gene, referred to as p-let-7d-luc. A similar vector with an 8-bp deletion of the predicted SMAD3-binding site is referred to as p-mlet-7d-luc. The luciferase scale is arbitrary and values represent averages of the triplicate assays. TGF-β stimulation was performed before DNA transfection. The dark blue columns represent the reporter construct without any stimulation, and the light blue columns represent the same with TGF-β stimulation.

Figure 3.

Tissue microarray analysis reveals that let-7d localizes within normal alveolar epithelium in control lungs and is nearly absent from fibrotic areas in idiopathic pulmonary fibrosis (IPF) lungs. (A) Panels i and iii, let-7d is localized in alveolar epithelial cells of control lungs, as evident by black staining (blue arrows); panels ii and iv, IPF lungs show almost a total absence of let-7d. The black arrows point to areas of dense fibrosis whereas blue arrows point to minimal staining for let-7d in the immediate surrounding areas. Scale bars: panels i and ii, 100 μm; panels iii and iv, 25 μm, in panel iii, 10 μm. (B) Number of let-7d–expressing cells per square millimeter was significantly lower in 40 slides from patients with IPF compared with 20 normal histology controls. *P < 0.001. (C) The forced vital capacity, expressed as a percentage of the predicted value (FVC%), of patients with IPF significantly increased (P = 2.1 × 10−6) with the number of let-7d–expressing cells per square millimeter. AECs = alveolar epithelial cells. Solid line connects the fitted values from a linear regression model. Dotted lines represent 95% confidence intervals on the fitted values.

Figure 4.

High-mobility group AT-hook 2 (HMGA2) localizes to alveolar epithelial cells in idiopathic pulmonary fibrosis (IPF) lungs and is regulated by transforming growth factor (TGF)-β and let-7 in vitro. (A) HMGA2 mRNA levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) in 10 control and 10 IPF lungs. (B) Immunolocalization of HMGA2 in IPF lungs (panels i, ii, and iii) and normal lungs (panel iv). Panels i and ii: Two different IPF lungs showing the immunoreactive protein, which was found primarily in alveolar epithelial cells, either in cytoplasm or nuclei (black arrows). The red arrows in panel i point to collapsed airspaces that are lined by HMGA2-positive epithelial cells. Panel iii: The same IPF lung as in panel ii, showing nuclear staining of HMGA2 in elongated epithelial cells (asterisk indicates an alveolar space) and some fibroblast-like cells immersed in a fibroblastic focus. Positive endothelial cells are marked with red arrows. Panel iv: Normal lungs were negative for HMGA2. Scale bars (all panels): 100 μm inset in panel (i) 100x. (C) HMGA2 mRNA levels determined by qRT-PCR in A549 cells at 0, 2, and 6 hours after stimulation with recombinant TGF-β (3 ng/ml). Results represent the average expression and SD of triplicate experiments. (D) HMGA2 mRNA levels determined by qRT-PCR in A549 cells 24 and 48 hours after transfection with 50 nM let-7d inhibitor. (E) HMGA2 mRNA levels in RLE-6TN cells 0, 2, and 6 hours after stimulation with recombinant TGF-β (5 ng/ml) after transfection with pre-let-7d or negative control 24 hours earlier.

Figure 5.

Inhibition of let-7d results in epithelial–mesenchymal transition changes. N-cadherin (CDH2), vimentin (VIM), and α-smooth muscle actin (ACTA2; α-SMA) mRNA levels were determined by quantitative real-time polymerase chain reaction in (A) A549 cells and (B) RLE-6TN cells 48 hours after transfection and in (C) NHBE cells 24 hours after transfection with 50 nM let-7d inhibitor. (D) Immunofluorescence imaging of A549 cells transfected with 50 nM let-7d inhibitor. Green fluorescence represents cytokeratin, an epithelial marker. Red fluorescence denotes the mesenchymal markers (CDH2, VIM, and ACTA2 [α-SMA]). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Whereas red staining was observed in cells transfected with let-7d inhibitor (right), there was no staining in cells transfected with a control oligonucleotide (left). (E) Western blots of RLE-6TN cells transfected with 50 nM let-7d inhibitor. scr = scrambled sequence; TJP1 = tight junction protein-1. (F) Densitometric analysis of the immunoblots in (E), *P < 0.05. Fold change by densitometry is equivalent to the density of the targeted protein divided by the density of the corresponding housekeeping gene. Results represent averages and SD of triplicate experiments.

Figure 6.

Effect of let-7 inhibition in vivo by intratracheal antagomir administration. Gene expression levels of (A) E-cadherin (CDH1), (B) tight junction protein-1 (TJP1 [ZO-1]), (C) collagen type 1A (COL1A), and (D) high-mobility group AT-hook 2 (HMGA2) levels in mice (n = 4) treated with intratracheal antagomir (10 mg/kg) or saline for 4 days. In both (E) and (F), panels i and iii are of saline-treated lungs and panels ii and iv are of antagomir-treated lungs. Scale bars: i and ii, 100 μm; iii and iv, 25 μm. (E) Masson trichrome staining after 18 days of saline or antagomir treatment. Arrows in ii and iv point to the blue staining for collagen. Note alveolar septal thickening. (F) Immunolocalization of α-smooth muscle actin (ACTA2). ACTA2 staining (brown) is significantly increased in antagomir-treated mice compared with saline-treated controls. (G) Quantitation of the total tissue in control and antagomir-treated lungs. Ten different fields (original magnification, ×20) from each slide were quantified, *P < 0.05. (H) Quantitation of collagen in control and antagomir-treated lungs. Ten different fields (original magnification, ×20) from each slide were quantified, *P < 0.05. (I) Number of ACTA2-expressing cells stained brown was significantly higher in antagomir-treated lungs in comparison with control lungs, *P < 0.05.

Figure 7.

Colocalization of α-smooth muscle actin (ACTA2) and S100A4 with SFTPC (surfactant, pulmonary-associated protein C). (A) The green fluorescence represents the mesenchymal markers ACTA2 and S100A4. The red fluorescence denotes the alveolar type II cell marker SFTPC. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. The merged image shows colocalization of S100A4 and SFTPC (top) and ACTA2 and SFTPC (bottom). The white arrows point to cells showing colocalization. The red arrows represent cells that express only SFTPC. (B) Relative quantitation of the colocalization of SFTPC (red) and ACTA2/ S100A4 (green) in scrambled control and antagomir-treated lungs. The results represent an average of four different fields per slide and three slides per group.