miR-9-5p suppresses pro-fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2

Abstract

Uncontrolled extracellular matrix (ECM) production by fibroblasts in response to injury contributes to fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Reactive oxygen species (ROS) generation is involved in the pathogenesis of IPF. Transforming growth factor-β1 (TGF-β1) stimulates the production of NADPH oxidase 4 (NOX4)-dependent ROS, promoting lung fibrosis (LF). Dysregulation of microRNAs (miRNAs) has been shown to contribute to LF. To identify miRNAs involved in redox regulation relevant for IPF, we performed arrays in human lung fibroblasts exposed to ROS. miR-9-5p was selected as the best candidate and we demonstrate its inhibitory effect on TGF-β receptor type II (TGFBR2) and NOX4 expression. Increased expression of miR-9-5p abrogates TGF-β1-dependent myofibroblast phenotypic transformation. In the mouse model of bleomycin-induced LF, miR-9-5p dramatically reduces fibrogenesis and inhibition of miR-9-5p and prevents its anti-fibrotic effect both in vitro and in vivo. In lung specimens from patients with IPF, high levels of miR-9-5p are found. In omentum-derived mesothelial cells (MCs) from patients subjected to peritoneal dialysis (PD), miR-9-5p also inhibits mesothelial to myofibroblast transformation. We propose that TGF-β1 induces miR-9-5p expression as a self-limiting homeostatic response.

Keywords: TGF‐β signaling; fibrosis; miRNAs; myofibroblast; oxidative stress.

Figure 1. miRNA expression data in human lung fibroblasts following stimulation with H₂O₂ or TGF‐β1

1. A

   Heat map showing relative miRNA expression between untreated (control) and H₂O₂‐treated HFL‐1 cells. The scale bar at the bottom left ranges from green to red (low to high expression) and numbers represent ΔC _t values. A blow‐up of differentially expressed miRNAs is depicted on the right side. Data are representative of results from two experiments performed independently.

2. B

   Volcano plot analysis of H₂O₂‐modulated miRNAs. Log₁₀ relative quantification (RQ) and negative (−) log₁₀ adjusted (adj.) P‐values are plotted on the x‐ and y‐axis, respectively. Each miRNA is represented by a colored dot, gray are down‐regulated, purple are up‐regulated and black are non‐regulated (adj. P‐value ≥ 0.05) miRNAs.

3. C–E

   qRT–PCR analysis of miR‐9‐5p expression in HFL‐1 cells pre‐incubated for 2 h either with polyethyleneglycol (PEG) or with 100 units/ml PEG‐catalase and treated with 100 μM H₂O₂ for the indicated times (n = 3) (C), in cells treated with 5 ng/ml TGF‐β1 for the indicated times (n = 3–8) (D) and in HFL‐1 cells pre‐incubated as described in (C) and treated with 5 ng/ml TGF‐β1 for 24 h (n = 4) (E). Bar graphs show mean ± SEM; two‐tailed Mann–Whitney U‐test (C, E) and Kruskal‐Wallis non‐parametric ANOVA (D); *P < 0.05, **P ˂ 0.01 compared to control cells, ^# P < 0.05 compared to the same time point of control cells and ^@ P < 0.05 compared to PEG‐catalase‐treated cells at time 0.

4. F

   Venn diagram showing the intersection of miR‐9‐5p targets and TGF‐β‐related genes.

Figure EV1. Effect of PEG‐catalase on H₂O₂‐ and TGF‐β1‐induced ROS production and effect of TGF‐β1 on the expression of miR‐9‐5p precursors and on ROS production in HFL‐1 cells

1. Lung fibroblasts were treated with 100 μM H₂O₂ for the indicated times after pre‐incubation for 2 h either with polyethylene glycol (PEG) or with 100 U/ml PEG‐catalase (n = 3–5). Intracellular ROS production was measured using 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) reagent and analyzed by FACS.

2. qRT–PCR analysis of miR‐9‐5p primary transcripts (pri‐miR‐9) levels in HFL‐1 cells stimulated with 5 ng/ml TGF‐β1 for the indicated times (n = 4–7).

3. Intracellular ROS production in HFL‐1 cells treated with 5 ng/ml TGF‐β1 for the indicated times was measured as described in (A) (n = 3–5).

4. Intracellular production of ROS in HFL‐1 cells pre‐treated as described above and treated with 5 ng/ml TGF‐β1 for 24 h was measured as described in (A) (n = 3–5).

Data information: Bar graphs show mean ± SEM; two‐tailed Mann–Whitney U‐test and Kruskal–Wallis non‐parametric ANOVA (B); *P < 0.05, **P < 0.01 compared to control cells and ^# P < 0.05 compared to control cells at the same time point. a.u., arbitrary units.

Figure 2. miR‐9‐5p regulates TGFBR2 and NOX4 expression in human lung fibroblasts

1. qRT–PCR analysis of TGFBR2 expression in HFL‐1 cells transfected with 40 nM pre‐miR‐NC (control) or pre‐miR‐9‐5p for 48 h (n = 5).

2. Western blot analysis (left) and quantification (n = 6) (right) of TGFBR2 protein levels in HFL‐1 cells transfected as indicated in (A).

3. Sequence of miR‐9‐5p and their base pairing (bars) with the BSs in the 3′ UTR of human TGFBR2 mRNA. PMs are symbolized by red letters.

4. Luciferase activity in HFL‐1 cells co‐transfected with psiCHECK2 containing WT or mutated 3′ UTR sequences of human TGFBR2 and 40 nM of pre‐miRs or miRNA inhibitors (n = 3).

5. qRT–PCR analysis of NOX4 expression in HFL‐1 cells transfected as described in (A) and treated with 5 ng/ml TGF‐β1 for the indicated times (n = 4).

6. Western blot analysis (above) and quantification (below) of NOX4 expression in HFL‐1 cells treated as described in (E) (n = 4).

7. Localization of the three miR‐9‐5p predicted BSs in the 3′ UTR of human NOX4 gene represented as described in (C).

8. Luciferase activity as described in (D) with the WT or mutated 3′ UTR sequences of human NOX4 gene (n = 3).

Data information: Data are shown as mean ± SEM; two‐tailed Mann–Whitney U‐test; and *P < 0.05, **P < 0.01 compared to control cells, ^# P < 0.05 compared to its corresponding negative control condition and ^## P < 0.01 compared to WT 3′ UTR sequence and pre‐miR‐9‐5p co‐transfected cells. a.u., arbitrary units; RLU, relative light units.

Figure 3. miR‐9‐5p inhibits TGF‐β1‐induced transformation of human lung fibroblasts into myofibroblasts

1. A

   qRT–PCR analysis of α‐SMA (n = 4), Col1α (n = 3) and FN (n = 3) expression levels in HFL‐1 cells transfected with 40 nM pre‐miR‐NC (control) or pre‐miR‐9‐5p and treated with 5 ng/ml TGF‐β1 for the indicated times.

2. B, C

   Protein levels (above) of α‐SMA (n = 4) (B) and FN (n = 3) (C) in HFL‐1 cells described in (A). Quantification of protein expression is shown below. a.u., arbitrary units.

3. D, E

   Fluorescence microscopy images of HFL‐1 cells stained with specific antibodies against α‐SMA (D, middle panels) and FN (E, middle panels) after transfection as described in (A) and TGF‐β1 treatment for 48 h (n = 3). Nuclei were stained with DAPI (blue). Scale bars: 100 μm.

4. F

   Bar graph represents percentage (%) of inhibitory effect of miR‐9‐5p on α‐SMA, Col1α1 and FN expression after over‐expression of TGFBR2, NOX4 or both. HFL‐1 cells were transfected with 3 μg pCMV5‐TGFBR2, 3 μg pCMV6‐NOX4 or 3 μg of each plasmid and 40 nM pre‐miR‐9‐5p and treated with 5 ng/ml TGF‐β1 for 24 h (n = 4). Control cells were transfected with 3 μg pCMV5 and 3 μg pCMV6.

Data information: All bar graphs show mean ± SEM; two‐tailed Mann–Whitney U‐test; *P < 0.05, **P < 0.01 compared to control cells and ^# P < 0.05, ^## P < 0.01 compared to its corresponding negative control time point.Source data are available online for this figure.

Figure EV2. Inhibition of miR‐9‐5p amplifies TGF‐β1‐induced transformation of human lung fibroblasts into myofibroblasts

1. A

   qRT–PCR analysis of Col1α and FN (n = 4) expression levels in HFL‐1 cells transfected with 40 nM miRNA inhibitor NC (control) or miRNA inhibitor‐9‐5p and treated with 5 ng/ml TGF‐β1 for 24 h.

2. B, C

   Protein levels (left) of α‐SMA (n = 5) (B) and FN (n = 4) (C) in HFL‐1 cells described in (A). Quantification of protein expression (right). a.u., arbitrary units.

3. D, E

   Fluorescence microscopy images of HFL‐1 cells stained with specific antibodies against α‐SMA (D, middle panels) and FN (E, middle panels) after transfection as described in (A) and TGF‐β1 treatment for 24 h (n = 3). Nuclei were stained with DAPI (blue). Scale bars: 100 μm.

Data information: All bar graphs show mean ± SEM; two‐tailed Mann–Whitney U‐test; *P < 0.05 , **P < 0.01 compared to control cells and ^# P < 0.05 compared to its corresponding negative control time point.

Figure 4. miR‐9‐5p inhibits the TGF‐β1‐induced fibrogenic pathway in human lung fibroblasts

1. Fluorescence images (left) and quantification (n = 3) (right) of migration in HFL‐1 cells transfected with either 40 nM pre‐miR‐NC (control) or pre‐miR‐9‐5p in response to TGF‐β1 for the indicated times. Nuclei were stained with DAPI (blue). Scale bar: 100 μm.

2. Fluorescence images (left) and quantification (n = 3) (right) of collagen matrix invasion in HFL‐1 cells transfected with either 40 nM pre‐miR‐NC or pre‐miR‐9‐5p in response to TGF‐β1 for 48 h. Nuclei were stained with DAPI (blue). Scale bar: 100 μm.

3. Luciferase activity of the reporter construct in HFL‐1 cells co‐transfected with 40 nM pre‐miR‐NC or pre‐miR‐9‐5p and treated with 5 ng/ml TGF‐β1 for 24 h (n = 3).

4. Western blot analysis (left) and quantification (n = 4) (right) of pSmad2 protein levels in HFL‐1 cells transfected as described in (A) and treated with 5 ng/ml TGF‐β1 for the indicated times. a.u., arbitrary units.

5. Fluorescence microscopy images of HFL‐1 cells stained with specific antibodies against Smad2/3 (green) after indicated treatments (n = 3). Nuclei were stained with DAPI (blue) and F‐actin was stained with phalloidin (red). Scale bar: 100 μm.

6. Bar graph represents percentage (%) of inhibitory effect of miR‐9‐5p on Smad2 phosphorylation after over‐expression of TGFBR2 in HFL‐1 cells transfected with 3 μg pCMV5‐TGFBR2 (TGFBR2) and 40 nM pre‐miR‐9‐5p and treated with 5 ng/ml TGF‐β1 for 15 min (densitometric analysis from four separate experiments). Control cells were transfected with 3 μg pCMV5.

Data information: Data are shown as mean ± SEM; two‐tailed Mann–Whitney U‐test; *P < 0.05 compared to control cells and ^# P < 0.05 compared to its corresponding negative control time point.

Figure 5. miR‐9‐5p is up‐regulated in lungs from both bleomycin‐treated mice and IPF patients

1. A, B

   qRT–PCR analysis of miR‐9‐5p (A), FN and Col1α1 (B) expression in lungs of mice after orotracheal bleomycin administration (1.5 U/kg body weight in 40 μl saline) for the indicated times (n = 6 mice per group). *P ˂ 0.05, **P ˂ 0.01 compared to day 0.

2. C

   Microphotographs of ISH showing miR‐9‐5p expression (purple, lower panels) in mouse lung samples at days 0 and 14 after bleomycin instillation (n = 3 mice per group). ISH with scrambled probes are shown in the upper images. Scale bar: 50 μm.

3. D

   qRT–PCR analysis of miR‐9‐5p expression levels in three histologically normal lungs (controls) and in seven lungs from IPF patients. *P ˂ 0.05 compared to control lungs.

Data information: Bar graphs show mean ± SEM; two‐tailed Mann–Whitney U‐test.

Figure EV3. Histopathological changes of lung tissue in mice treated with bleomycin

Microphotographs of H&E (upper panels) and Masson's trichrome (bottom panels) staining of lung tissue sections from saline‐treated control (day 0) as well as from orotracheally bleomycin‐treated mice (1.5 U/kg body weight in 40 μl saline serum) for the indicated times (n = 3 mice per group). H&E showed intact alveoli and normal interstitium in lungs of control mice and gradually increased areas of alveolar and interstitial fibrosis (black arrows), lung alveoli destruction and interstitium thickening in lungs of bleomycin‐treated mice. Intense blue Masson's staining indicates regions with collagen deposition. Scale bar: 100 μm.

Figure EV4. Histological characterization of human lung tissue of IPF patients

1. A, B

   Microphotographs of H&E (upper panel in each case) and Masson's trichrome (bottom panel in each case) staining in paraffin‐embedded lung tissue sections from two histologically normal lungs (controls) (A) and seven IPF patients (B). As it can be observed in (A) control lung tissue is characterized by a thin pulmonary alveolar epithelium. Neither fibroblast nor ECM deposits are present in the interstitium. In IPF patients (B), an extensive pulmonary fibrosis was observed in all the specimens analyzed. Alveolar interstitium is fully occupied by fibroblasts, inflammatory infiltrates and markedly visible ECM deposits (blue in Masson's staining for collagen). Alveolar epithelium is barely observed due to fibrosis development. All images are shown at equal magnification. Scale bar: 100 μm.

Figure EV5. Determination of miR‐9‐5p delivery and quantification of miR‐9‐5p expression level after lentiviral vector infection

1. GFP‐expressing fluorescence images of frozen lung sections from mice orotracheally instilled with lenti‐SC (control) or lenti‐miR‐9 vector (1 × 10⁶ i.f.u./mouse) for the indicated days (n = 3 mice in each group). Scale bar: 100 μm.

2. qRT–PCR analysis of miR‐9‐5p expression in lungs from mice after 4 days of lentiviral infection (n = 6 mice per group).

3. qRT–PCR analysis of Col1α1 and FN in lungs from mice administered saline or lenti‐SC 4 days before orotracheal bleomycin instillation (1.5 U/kg body weight in 40 μl saline serum) and sacrificed 14 days after treatment (n = 3 mice per group).

4. qRT–PCR analysis of miR‐9‐5p expression in lungs from mice administered miRNA inhibitor NC (control) or miRNA inhibitor‐9‐5p (7 mg/kg body weight in 40 μl saline). The miRNA inhibitor was instilled at days −4 and −2 before sacrifice (n = 4 mice per group).

Data information: All bar graphs show mean ± SEM; two‐tailed Mann–Whitney U‐test; *P < 0.05, **P < 0.01 compared to its corresponding negative control condition; no significant differences between saline and lenti‐SC‐treated mice were found.

Figure 6. miR‐9‐5p prevents pulmonary fibrosis in mice

1. A

   qRT–PCR analysis of α‐SMA, Col1α1 and FN expression in lungs from mice administered 1 × 10⁶ i.f.u. of lenti‐SC (control) or lenti‐miR‐9 for 4 days followed by orotracheal bleomycin administration (1.5 U/kg body weight in 40 μl saline) or saline for 14 days (n = 4 mice per group).

2. B

   Microphotographs of H&E (upper panels) and Masson's trichrome staining (middle panels) and α‐SMA expression (lower panels) from lung sections of mice treated as described in (A) (n = 4 mice per group). Scale bars: 100 μm.

3. C, D

   Semiquantitative determination (grade 0 to 3) of the collagen content (C) and the quantity of myofibroblasts (D) in lung tissue samples from mice treated as described in (A) (n = 5 mice per group). Each mouse is represented by a symbol, dots represent lenti‐SC and squares represent lenti‐miR‐9‐treated mice, respectively.

4. E

   Total area of lung fibrosis (mm²) in lung sections from mice treated as described in (A) (n = 4 mice per group).

Data information: Data are shown as median ± SEM; two‐tailed Mann–Whitney U‐test; *P ˂ 0.05 compared to mice given control lentivirus and saline‐treated and ^# P < 0.05 compared to mice given control lentivirus and bleomycin‐treated.

Figure 7. miR‐9‐5p regulates TGFBR2 and NOX4 expression and inhibits Smad2 phosphorylation in mouse lungs

1. qRT–PCR analysis of TGFBR2 expression in lungs from mice administered lenti‐SC (control) or lenti‐miR‐9 (1 × 10⁶ i.f.u.) (n = 4 mice per group).

2. qRT–PCR analysis of NOX4 expression in lungs from mice given lenti‐SC (control) or lenti‐miR‐9 (1 × 10⁶ i.f.u. per mouse) for 4 days followed by orotracheal bleomycin administration (1.5 U/kg body weight in 40 μl saline) or saline for 14 days (n = 4 mice per group).

3. Microphotographs of TGFBR2 and pSmad2 expression in mouse lung samples described in (B) (n = 4 mice per group). Scale bars: 100 μm.

Data information: Bar graphs show mean ± SEM; two‐tailed Mann–Whitney U‐test; *P ˂ 0.05 compared to saline‐treated mice administered lenti‐SC and ^# P ˂ 0.05 compared to bleomycin‐treated mice administered lenti‐SC.

Figure 8. miR‐9‐5p inhibition exacerbates pulmonary fibrosis in mice

1. A

   qRT–PCR analysis of Col1α1 and FN expression in lungs from mice administered miRNA inhibitor NC (control) or miRNA inhibitor‐9‐5p (7 mg/kg body weight in 40 μl saline) 4 and 2 days before orotracheal instillation of bleomycin (1.5 U/kg body weight in 40 μl saline) or saline for 10 days (n = 3–6 mice per group).

2. B

   Microphotographs of H&E (upper panels) and Masson's trichrome staining (middle panels) and α‐SMA expression (lower panels) from lung sections of mice treated as described in (A) (n = 3–7 mice per group). Scale bars: 100 μm.

3. C, D

   Semiquantitative determination (grade 0 to 3) of the collagen content (C) and the quantity of myofibroblasts (D) in lung tissue samples from mice treated as described in (A) (n = 5–7 mice per group). Each mouse is represented by a symbol, dots represent miRNA inhibitor NC and squares represent miRNA inhibitor‐9‐5p‐treated mice, respectively.

Data information: Data are shown as mean ± SEM; two‐tailed Mann–Whitney U‐test; *P ˂ 0.05, **P ˂ 0.01 compared to mice given control miRNA inhibitor and saline‐treated, and ^# P < 0.05, ^## P < 0.01 compared to mice given control miRNA inhibitor and bleomycin‐treated.

Figure 9. miR‐9‐5p is up‐regulated in non‐epith effluent‐derived MCs from PD patients, attenuates TGF‐β1‐induced fibrogenesis and regulates TGFBR2 and NOX4 expression in omentum‐derived MCs

1. qRT–PCR analysis of miR‐9‐5p expression in omentum‐derived MCs (n = 3) epith (n = 5) and non‐epith (n = 4) effluent‐derived MCs. *P ˂ 0.05 compared to omentum‐derived MCs and ^# P ˂ 0.05 compared to epith effluent‐derived MCs.

2. qRT–PCR analysis of miR‐9‐5p expression in omentum‐derived MCs treated with 5 ng/ml TGF‐β1 for 48 h (n = 3). *P ˂ 0.05 compared to untreated cells.

3. qRT–PCR analysis of α‐SMA, Col1α1 and FN in omentum‐derived MCs transfected with 40 nM pre‐miR‐NC (control) or pre‐miR‐9‐5p and treated with 5 ng/ml TGF‐β1 for 48 h (n = 3). *P < 0.05 compared to negative control‐transfected cells and ^# P < 0.05 compared to the corresponding negative control time point.

4. Western blot analysis (left) and quantification (right) of α‐SMA protein levels in cells treated as described in (C) (n = 3). a.u., arbitrary units. *P < 0.05 compared to negative control‐transfected cells and ^# P < 0.05 compared to the corresponding negative control time point.

5. Fluorescence images (left) and quantification (right) of collagen matrix invasion in omentum‐derived MCs transfected with either 40 nM pre‐miR‐NC or pre‐miR‐9‐5p in response to TGF‐β1 for 24 h (n = 3). Nuclei were stained with DAPI (blue). Scale bar: 100 μm.

6. qRT–PCR analysis of TGFBR2 expression in omentum‐derived MCs transfected with 40 nM of pre‐miR‐NC (control) or pre‐miR‐9‐5p for 48 h (n = 3).

7. qRT–PCR analysis of NOX4 expression in omentum‐derived MCs treated as described in (C) (n = 3).

Data information: Bar graphs show mean ± SEM; two‐tailed Mann–Whitney U‐test; *P < 0.05 compared to negative control‐transfected cells and ^# P < 0.05 compared to the corresponding negative control time point.

Figure 10. Regulation of fibrogenesis by miR‐9‐5p

ROS and TGF‐β1 induce miR‐9‐5p expression. Over‐expression of miR‐9‐5p in lung fibroblasts and omentum‐derived MCs blocks TGFBR2 and NOX4 expression, thus preventing myofibroblast differentiation, ECM deposition and organ fibrogenesis.