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. 2020 Jul 21;11(7):553.
doi: 10.1038/s41419-020-02747-9.

CircRNA TADA2A relieves idiopathic pulmonary fibrosis by inhibiting proliferation and activation of fibroblasts

Juan Li  1 , Ping Li  1 , Guojun Zhang  1 , Pan Qin  2 , Da Zhang  2 , Wei Zhao  3
Affiliations

CircRNA TADA2A relieves idiopathic pulmonary fibrosis by inhibiting proliferation and activation of fibroblasts

Juan Li et al. Cell Death Dis. .

Abstract

The excessive activation and proliferation of lung fibroblasts are responsible for the abundant deposition of extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF), while its specific mechanism is still unknown. This study focuses on the role of circRNA (circ) TADA2A in functional abnormalities of lung fibroblasts and aims to elaborate its regulatory mechanism. In the present study, circTADA2A was downregulated in both IPF primary human lung fibroblasts and human IPF fibroblastic cell lines. Functionally, the overexpression of circTADA2A repressed the activation and proliferation of normal human fibroblastic cell line induced by several fibrogenic growth factors. Using fluorescence in situ hybridization (FISH), luciferase reporter assays, and RNA pull-down, circTADA2A was confirmed to function as sponges of miR-526b and miR-203, thus releasing the expression of Caveolin (Cav)-1 and Cav2. The overexpression of circTADA2A suppressed lung-fibroblasts activation via Cav1 and reduced lung-fibroblasts proliferation via Cav2. In vivo experiments also confirmed that the overexpression of circTADA2A decreased fibrogenic responses induced by bleomycin in lung-fibrosis mice. Collectively, circTADA2A repressed lung-fibroblasts activation via miR-526b/Cav1 and reduced lung-fibroblasts proliferation via miR-203/Cav2, thus inhibiting the excessive deposition of ECM and relieving IPF.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. CircTADA2A was downregulated in IPF lung fibroblasts.
a Circular RNAs (circRNAs) expression levels in IPF primary human lung fibroblasts (IPF-HLF; n = 5) and normal primary human lung fibroblasts (N-HLF; n = 5) were measured by qRT-PCR. *P < 0.05, **P < 0.01 vs N-HLF. b Left: CircTADA2A expression in human IPF fibroblastic cell lines (LL-97A and LL-29) and normal human fibroblastic cell line (LL-24) was measured by qRT-PCR. Right: Representative fluorescence in situ hybridization (FISH) images of circTADA2A in LL-97A, LL-29, and LL-24 (blue, DAPI; red spot, positive staining; Scale bar = 20 µm). **P < 0.01 vs LL-24 cells. c LL-24 cells were stimulated with fetal calf serum (FCS; 2% or 5%), platelet-derived growth factor-BB (PDGF-BB; 30 or 60 ng/ml), insulin-like growth factor 1 (IGF-1; 100 or 200 ng/ml), and transforming growth factor-β1 (TGF-β1; 5 or 10 ng/ml) for 6 h, respectively. CircTADA2A expression was measured by qRT-PCR. *P < 0.05, **P < 0.01 vs LL-24 cells without stimulation.
Fig. 2
Fig. 2. CircTADA2A inhibited growth-factors-driven fibroblasts proliferation and activation.
LL-24 cells were transfected with a si-circTADA2A or its negative control (si-control) or b adenovirus vector (Ad)-circTADA2A or its negative control (Ad-GFP). Cell proliferation was measured by BrdU incorporation assay after stimulation of 5% FCS or 60 ng/ml PDGF-BB or 200 ng/ml IGF-1 for 6 h. The protein expression of collagen 1a1 (COL1A1), collagen 3a1 (COL3A1), laminin (LN), fibronectin (FN), and α-smooth muscle actin (α-SMA) were determined by western blot after stimulation of 10 ng/ml TGF-β1 for 6 h, GAPDH was used as an internal control. *P < 0.05, **P < 0.01.
Fig. 3
Fig. 3. CircTADA2A bound to miR-203 and miR-526b.
a LL-29 cells were transfected with Ad-circTADA2A or Ad-GFP, the expression levels of miR-203, miR-520f, miR-450b-3p, miR-526b, miR-638, and miR-769-3p were measured by qRT-PCR. **P < 0.01 vs Ad-GFP. b LL-24 cells were transfected with si-circTADA2A or si-control, the expression levels of miR-526b and miR-203 were measured by qRT-PCR. **P < 0.01 vs si-control. c HEK 293 T (293 T) cells were transfected with miR-526b mimic or miR-203 mimic or miR-526b mimic+miR-203 mimic or negative control (Pre-NC), relative luciferase activities of circTADA2A vector were measured using Dual-Luciferase Reporter Assay System. *P < 0.05, **P < 0.01 vs Pre-NC. d Detection of miR-526b and miR-203 using qRT-PCR in the samples pulled down by the circTADA2A probe and its negative control (Oligo probe). **P < 0.01 vs Oligo probe. e Colocalization between circTADA2A (red) and miR-526b/miR-203 (green) was observed (arrowheads) using FISH in LL-29 cells. The nuclei were stained with DAPI. Scale bar = 20 µm.
Fig. 4
Fig. 4. CircTADA2A released Cav1 and Cav2 expressions via targeting miR-526b and miR-203.
a Putative binding sites between miR-526b and Cav1 were forecasted by Miranda. The expression of Cav1 was determined by western blot in LL-29 cells that have been transfected with miR-526b inhibitor or its negative control (NC). Luciferase activity of Cav1-3′-UTR wide type (WT) and Cav1-3′-UTR mutation (MUT) in 293 T cells that have been transfected with miR-526b inhibitor or NC. The expression of Cav1 was determined by western blot in b LL-29 cells that have been transfected with Ad-circTADA2A, miR-526b mimic, or the negative control of miR-526b mimic (Pre-NC), and c LL-24 cells that have been transfected with si-circTADA2A, miR-526b inhibitor, or NC. GAPDH was used as an internal control. d Putative binding sites between miR-203 and Cav2. The expression of Cav2 and the luciferase activity of Cav2-3′-UTR WT and Cav2-3′-UTR MUT were determined. The expression of Cav2 was determined by western blot in e LL-29 cells that have been transfected with Ad-circTADA2A, miR-203 mimic, or Pre-NC, and f LL-24 cells that have been transfected with si-circTADA2A, miR-203 inhibitor, or NC. GAPDH was used as an internal control. **P < 0.01 vs NC.
Fig. 5
Fig. 5. CircTADA2A inactivated lung fibroblasts via Cav1.
LL-24 cells were transfected with a si-circTADA2A or Ad-Cav1+si-circTADA2A or Ad-GFP + si-circTADA2A or b Ad-circTADA2A or Ad-GFP or Ad-circTADA2A+si-control or Ad-circTADA2A+si-Cav1, and then stimulation with 5% FCS or 60 ng/ml PDGF-BB or 200 ng/ml IGF-1 or 10 ng/ml TGF-β1 for 6 h. The expression of COL1A1, COL3A1, LN, FN, and α-SMA were measured by western blot, and the cell proliferation was measured by BrdU incorporation assay. **P < 0.01.
Fig. 6
Fig. 6. CircTADA2A inhibited lung-fibroblasts proliferation via Cav2.
LL-24 cells were transfected with a si-circTADA2A or si-circTADA2A+Ad-Cav2 or si-circTADA2A+Ad-GFP or b Ad-circTADA2A or Ad-GFP or Ad-circTADA2A+si-control or Ad-circTADA2A+si-Cav2, and then stimulation with 5% FCS or 60 ng/ml PDGF-BB or 200 ng/ml IGF-1 or 10 ng/ml TGF-β1 for 6 h. The expression of COL1A1, COL3A1, LN, FN, and α-SMA were measured by western blot, and the cell proliferation was measured by BrdU incorporation assay. **P < 0.01.
Fig. 7
Fig. 7. CircTADA2A alleviated pulmonary fibrosis in lung-fibrosis mice.
Mice were divided into four groups: saline (n = 7), bleomycin (BLM; n = 7), BLM + adenovirus circTADA2A (circTADA2A; n = 7), and BLM + adenovirus vector (vector; n =7). Mice were infected with Ad-circTADA2A or Ad-vector, 2 days later, BLM (3 U/kg) in 50 μl saline was administered intratracheally in mice to induce pulmonary fibrosis. a Two weeks after BLM stimulation, lung-function measurements of mice (total lung capacity, lung compliance, and tissue resistance) were measured, and then the mice were sacrificed and the lung tissues were collected for the following experiments. b H&E and Masson staining of each group. Scale bars = 200 μm. c Hydroxyproline content of lung homogenates was evaluated using a Hydroxyproline Assay Kit. d The expression level of circTADA2A was measured by qRT-PCR. e The expression levels of miR-526b and miR-203 were measured by qRT-PCR. f The expression levels of Cav1 and Cav2 were measured by western blot. GAPDH was used as an internal control. **P < 0.01.
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