lncRNA ZEB1-AS1 promotes pulmonary fibrosis through ZEB1-mediated epithelial-mesenchymal transition by competitively binding miR-141-3p

Abstract

Long non-coding RNAs (lncRNAs) have been reported to be involved in various pathophysiological processes in many diseases. However, the role and mechanism of lncRNAs in pulmonary fibrosis have not been explicitly delineated. In the present study, we found that lncRNA ZEB1 antisense RNA 1 (ZEB1-AS1) is upregulated in the lungs of BLM-induced rats and TGF-β1-induced RLE-6TN cells, and positively correlated with the levels of ZEB1, an epithelial-mesenchymal transition (EMT) master regulator. Knockdown of ZEB1-AS1 alleviated BLM-induced fibrogenesis, in vivo, via inhibiting EMT progress. Mechanistically, we identified that ZEB1-AS1 promoted fibrogenesis in RLE-6TN cells and ZEB1-AS1 silencing inhibited TGF-β1-induced fibrogenesis through modulation of miR-141-3p. Further experiments revealed that ZEB1-AS1 acted as competing endogenous RNA (ceRNA) of miR-141-3p: forced expression of ZEB1-AS1 reduced the expression of miR-141-3p to activate Zinc-finger Ebox Binding Homeobox 1 (ZEB1) in RLE-6TN cells. In addition, we found that upregulation of miR-141-3p prevented fibrogenesis by targeting ZEB1. Therefore, our finding suggested lncRNA ZEB1-AS1 as a new profibrotic molecule that acts as a regulator of miR-141-3p/ZEB1 axis during lung fibrosis and demonstrated ZEB1-AS1 as a potential therapeutic target for the prevention and treatment of pulmonary fibrosis.

Fig. 1. Upregulated ZEB1-AS1 in pulmonary fibrosis is positively correlated with ZEB1 expression.

RT-qPCR was carried out to determine the relative expression of ZEB1-AS1 (a) and ZEB1 (b) mRNA in BLM-induced lung tissues (IPF group, n = 10) and normal lung tissues (Normal group, n = 10). Spearman analysis was used to analyze the association between ZEB1-AS1 and ZEB1 expression in the lung tissues from Normal group (c) and IPF group (d). Relative expression of ZEB1-AS1 (e) and ZEB1 (f) mRNA in RLE-6TN cells treated with 10 ng/ml TGF-β1 for 48 h, as detected by RT-qPCR. g RNA FISH was performed to determine the location of endogenous ZEB1-AS1 (red) in RLE-6TN cells. U6 and GAPDH were used as nuclear and cytoplasmic localization markers, respectively. DNA (blue) was stained with DAPI. h Nucleocytoplasmic separation result confirmed that ZEB1-AS1 was mainly expressed in the cytoplasm by using RT-qPCR. *P < 0.05, **P < 0.01

Fig. 2. Knockdown of lncRNA ZEB1-AS1 inhibits bleomycin (BLM)-induced pulmonary fibrosis, in vivo.

a RT-qPCR analysis showed the expression of ZEB1-AS1 in the lungs from normal rats and rats treated with adenovirus associated virus 5 (AAV5) carrying sh-ZEB1-AS1 (n = 10). b Treatment with AAV5 carrying sh-ZEB1-AS1 showed decreased ZEB1-AS1 expression compared with those in the BLM group as confirmed by RT-qPCR analysis (n = 10). c H&E and Masson’s trichrome staining indicated the collagen deposition, and the areas of fibrosis in rats with or without knockdown of ZEB1-AS1; IHC analysis showed the expression of levels of collagen 1 and fibronectin 1 (FN1) in lung tissues after knockdown of ZEB1-AS1. d Double-labeling immunofluorescence demonstrated the overlap of E-cadherin (red) and a-SMA (green) in lung tissues after knockdown of ZEB1-AS1. e Western blot analysis showed the relative expression of EMT-related markers, α-SMA, E-cadherin, Collagen 1, and FN1 protein were detected in indicated groups (means ± SD, n = 3). *P < 0.05, **P < 0.01

Fig. 3. lncRNA ZEB1-AS1 contributes to fibrogenesis in alveolar type II epithelial cells by inhibiting the function of miR-141-3p.

a, b EdU incorporation assay was used to assess the effect of ZEB1-AS1 on cells proliferation in RLE-6TN with or without upregulation of miR-141-3p. Scale bar = 100 μm. c, d Wound-healing assay reveals the effect of ZEB1-AS1 on cell migration in RLE-6TN cells with or without upregulation of miR-141-3p. Scale bar = 200 μm. RT-qPCR was performed to measure the expression of Col 1α1 (e) and Col 3α1 (f) in RLE-6TN cells following co-transfection of pcDNA3.1 or ZEB1-AS1 vector, miR-141-3p or miR-NC. g Representative images of immunofluorescence staining in each group to determine protein expression of E-cadherin (red) and a-SMA (green) accompanied with nuclei stained by DAPI (blue) in RLE-6TN cells in groups, as indicated. h, i Western blot analysis was performed to measure the expression of epithelial–mesenchymal transition (EMT)-related proteins α-SMA, E-cadherin, Collagen 1, and Fibronectin 1 (FN1), and β-actin was used as a loading control (means ± SD, n = 3). *P < 0.05, **P < 0.01

Fig. 4. Knockdown of ZEB1-AS1 alleviates TGF-β1-induced fibrogenesis in alveolar type II epithelial cells through miR-141-3p regulation.

a, b EdU incorporation assay was performed to assess the effect of ZEB1-AS1 depletion on cells proliferation in TGF-β1-induced RLE-6TN cells. Scale bar = 100 μm. c, d Wound-healing assay revealed the effect of ZEB1-AS1 migration ability in TGF-β1-induced RLE-6TN cells with or without downregulation of miR-141-3p. Scale bar = 200 μm. RT-qPCR was used to measure the expression of Col 1α1 (e) and Col 3α1 (f) mRNA in TGF-β1-treated RLE-6TN cells with ZEB1-AS1/miR-141-3p downregulation. g Double-labeling immunofluorescence demonstrated the protein expression of E-cadherin (red) and a-SMA (green) accompanied with nuclei stained by DAPI (blue) in RLE-6TN cells. h, i Western blot analysis showed the expression of epithelial–mesenchymal transition (EMT)-related proteins α-SMA, E-cadherin, Collagen 1, and Fibronectin 1 (FN1), and β-actin was used as a loading control (means ± SD, n = 3). *P < 0.05, **P < 0.01

Fig. 5. lncRNA ZEB1-AS1 regulates ZEB1 expression by sponging miR-141-3p.

a RT-qPCR was used to determine the expression of miR-141-3p in BLM-induced lung tissues (IPF group, n = 10) and normal lung tissues (Normal group, n = 10). b Relative expression of miR-141-3p in the lung tissues with or without ZBE1-AS1 depletion. c Relative expression of miR-141-3p in RLE-6TN cells treated with 10 ng/ml TGF-β1 for 48 h. d Relative expression of miR-141-3p in RLE-6TN cells with or without knockdown of ZEB1-AS1. e Bioinformatics predicted the binding sites between ZEB1-AS1 and miR-141-3p and the schematic diagram shows the sequences of ZEB1-AS1 3ʹ-UTR wild-type and mutant with miR-141-3p. f The dual-luciferase reporter gene assay was performed to identify the interaction between ZEB1-AS1 and miR-141-3p. Luciferase activities were calculated as the ratio of firefly/renilla activities and normalized to the miR-NC + ZEB1-AS1 wile-type group. g ZEB1-AS1 decreased miR-141-3p activity. RLE-6TN cells were infected with the miR-141-3p sensor and then transfected with ZEB1-AS1 or sh-ZEB1-AS1. Luciferase activity of the miR-141-3p sensor was increased in cells treated with ZEB1-AS1, whereas decreased after knockdown of ZEB1-AS1. h ZEB1-AS1 acts as a sponge for miR-141-3p. RLE-6TN cells were infected with ZEB1-AS1 and pcDNA3.1 followed by transfection with the miR-141-3p sensor. ZEB1-AS1 ablated the inhibitory effects of miR-141-3p on its sensor, as determined by the luciferase assay. *P < 0.05, **P < 0.01

Fig. 6. ZEB1-AS1/miR-141-3p downregulates the expression of ZEB1 in cultured alveolar type II epithelial cells.

a Spearman analysis was used to analyze the association between miR-141-3p and ZEB1 expression; r = −0.704, P = 0.016. b The seed sequences of miR-141-3p match the 5ʹUTR of ZEB1, as predicted by starBase v3.0 (http://starbase.sysu.edu.cn/). c The 293T cells were co-transfected with 100 nM miR-141-3p or miRNA negative control (miR-NC) and the luciferase constructs carrying ZEB1 5ʹUTR. Luciferase assay was performed 24 h after transfection. d Western blot analysis and e RT-qPCR were performed to measure the expression of ZEB1 in ZEB1-AS1/miR-141-3p upregulated groups. *P < 0.05, **P < 0.01

Fig. 7. Overexpression of miR-141-3p alleviates TGF-β1-driven fibrogenesis in alveolar type II epithelial cells by targeting ZEB1.

a, b EdU incorporation assay was used to assess the effect of miR-41-3p cells proliferation in TGF-β1-induced RLE-6TN cells. Scale bar = 100 μm. c, d Wound-healing assay reveals the effect of miR-41-3p on cell migration in TGF-β1-induced RLE-6TN cells. Scale bar = 200 μm. RT-qPCR was performed to detect the expression of Col 1α1 (e) and Col 3α1 (f) mRNA in TGF-β1-treated RLE-6TN cells following co-transfection of miR-141-3p, miR-NC, pcDNA3.1, or ZEB1 vector. g Double-labeling immunofluorescence demonstrated the overlap of E-cadherin (red) and a-SMA (green) in RLE-6TN cells in groups, described above. h, i Relative expression of EMT-related markers, α-SMA, E-cadherin, Collagen 1, and FN1 protein were detected in indicated groups through western blot analysis (means ± SD, n = 3). *P < 0.05, **P < 0.01