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. 2021 Aug;25(15):7294-7306.
doi: 10.1111/jcmm.16758. Epub 2021 Jun 27.

LncRNA-ATB regulates epithelial-mesenchymal transition progression in pulmonary fibrosis via sponging miR-29b-2-5p and miR-34c-3p

Qi Xu  1 , Demin Cheng  1 , Yi Liu  1 , Honghong Pan  1 , Guanru Li  1 , Ping Li  1 , Yan Li  1 , Wenqing Sun  1 , Dongyu Ma  1 , Chunhui Ni  1
Affiliations

LncRNA-ATB regulates epithelial-mesenchymal transition progression in pulmonary fibrosis via sponging miR-29b-2-5p and miR-34c-3p

Qi Xu et al. J Cell Mol Med. 2021 Aug.

Abstract

Dysregulation of non-coding RNAs (ncRNAs) has been proved to play pivotal roles in epithelial-mesenchymal transition (EMT) and fibrosis. We have previously demonstrated the crucial function of long non-coding RNA (lncRNA) ATB in silica-induced pulmonary fibrosis-related EMT progression. However, the underlying molecular mechanism has not been fully elucidated. Here, we verified miR-29b-2-5p and miR-34c-3p as two vital downstream targets of lncRNA-ATB. As opposed to lncRNA-ATB, a significant reduction of both miR-29b-2-5p and miR-34c-3p was observed in lung epithelial cells treated with TGF-β1 and a murine silicosis model. Overexpression miR-29b-2-5p or miR-34c-3p inhibited EMT process and abrogated the pro-fibrotic effects of lncRNA-ATB in vitro. Further, the ectopic expression of miR-29b-2-5p and miR-34c-3p with chemotherapy attenuated silica-induced pulmonary fibrosis in vivo. Mechanistically, TGF-β1-induced lncRNA-ATB accelerated EMT as a sponge of miR-29b-2-5p and miR-34c-3p and shared miRNA response elements with MEKK2 and NOTCH2, thus relieving these two molecules from miRNA-mediated translational repression. Interestingly, the co-transfection of miR-29b-2-5p and miR-34c-3p showed a synergistic suppression effect on EMT in vitro. Furthermore, the co-expression of these two miRNAs by using adeno-associated virus (AAV) better alleviated silica-induced fibrogenesis than single miRNA. Approaches aiming at lncRNA-ATB and its downstream effectors may represent new effective therapeutic strategies in pulmonary fibrosis.

Keywords: EMT; ceRNA; lncRNA-ATB; pulmonary fibrosis.

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Conflict of interest statement

The authors confirm that there are no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
LncRNA‐ATB acts as a sponge of miR‐29b‐2‐5p and miR‐34c‐3p. A, qRT‐PCR analysis of lncRNA‐ATB, miR‐29b‐2‐5p and miR‐34c‐3p expression in A549 cells (mean ± SD, n = 3), with *P < .05 vs the control group and # P < .05 vs the TGF‐β1‐treated group. B, Schematic diagram of target sites of miR‐29b‐2‐5p and miR‐34c‐3p in the lncRNA‐ATB. C, Luciferase reporter assays of relative luciferase activity of in A549 transfected with ATB‐01‐wt/ATB‐01‐mut and ATB‐02‐wt/ATB‐02‐mut (mean ± SD, n = 3), with **P < .01 vs the miR‐29b‐2‐5p NC‐mimic group. D, Luciferase reporter assays of relative luciferase activity of in A549 transfected with ATB‐03/04‐wt, ATB‐03‐mut, ATB‐04‐mut and ATB‐03/04‐mut (mean ± SD, n = 3), with **P < .01 vs the miR‐34c‐3p NC‐mimic group. E, Luciferase reporter assays of relative luciferase activity of in A549 transfected with ATB‐05‐wt and ATB‐05‐mut (mean ± SD, n = 3). F, miR‐29b‐2‐5p, miR‐34c‐3p and lncRNA‐ATB enrichment as determined from RIP assays performed using IgG or AGO2 antibodies, followed by qRT‐PCR (mean ± SD, n = 3), **P < .01. G, The relative levels of lncRNA‐ATB in A549 cells were pulled down by biotinylated miR‐29b‐2‐5p or miR‐34c‐3p (mean ± SD, n = 3), with **P < .01 vs Bio‐miR‐NC group
FIGURE 2
FIGURE 2
TGF‐β1 promotes EMT process and decreases miR‐29b‐2‐5p and miR‐34c‐3p levels. A, Western blot analysis of E‐cadherin, Vimentin, Fibronectin and α‐SMA in A549 cells and BEAS‐2B cells treated with 0, 1, 2 and 5 ng/mL TGF‐β1 for 48 h. B, Western blot analysis of E‐cadherin, Vimentin, Fibronectin and α‐SMA in human primary type II AECs treated with 0 or 5 ng/mL TGF‐β1 for 48 h. C, Wound healing assays were performed to measure the migration ability of A549 cells treated with 0, 1, 2 and 5 ng/mL TGF‐β1 for 48 h. D, Immunofluorescence staining of Vimentin and E‐cadherin in A549 cells for the control and TGF‐β1 (0 or 5 ng/mL) treatment groups. Green represents Vimentin staining; red represents E‐cadherin staining; and blue represents nuclear DNA staining by DAPI. The scale bar is 50 μm. E, qRT‐PCR detection of lncRNA‐ATB expression in 0, 1, 2 and 5 ng/mL TGF‐β1 treated A549 and BEAS‐2B cells for 48 h (mean ± SD, n = 3), *P < .05 difference from untreated cells. F, qRT‐PCR analysis of miR‐29b‐2‐5p and miR‐34c‐3p expression in 0, 1, 2 and 5 ng/mL TGF‐β1 treated A549 and BEAS‐2B cells for 48 h (mean ± SD, n = 3), *P < .05 difference from untreated cells. G, qRT‐PCR detection of lncRNA‐ATB, miR‐29b‐2‐5p and miR‐34c‐3p expression in human primary type II AECs after 0 or 5 ng/mL TGF‐β1 treatment (mean ± SD, n = 3), *P < .05
FIGURE 3
FIGURE 3
MiR‐29b‐2‐5p and miR‐34c‐3p mediate the function of lncRNA‐ATB in regulating EMT. A, The miR‐29b‐2‐5p and miR‐34c‐3p were detected by qRT‐PCR in A549 and BEAS‐2B cells transfected with miR‐29b‐2‐5p mimic, miR‐34c‐3p mimic or NC‐mimic (mean ± SD, n = 3), **P < .01. B, Western blot detected levels of E‐cadherin, Vimentin, Fibronectin and α‐SMA in A549 cells transfected with miR‐29b‐2‐5p or miR‐34c‐3p mimic then treated with 5 ng/mL TGF‐β1 for 48 h. C, Cell migration was measured using a wound healing assay in A549 cells transfected with miR‐29b‐2‐5p or miR‐34c‐3p mimic then treated with 5 ng/mL TGF‐β1 for 48 h (mean ± SD, n = 3), **P < .05 difference from untreated cells. D, The expression of Vimentin was detected by immunofluorescence staining in A549 cells transfected with miR‐29b‐2‐5p or miR‐34c‐3p mimic then treated with 5 ng/mL TGF‐β1 for 48 h. E‐F, Western blot of the protein expression of E‐cadherin, Vimentin, Fibronectin and α‐SMA in treated A549 cells for the indicated groups
FIGURE 4
FIGURE 4
MEKK2 and NOTCH2 are two functional downstream targets of miR‐29b‐2‐5p and miR‐34c‐3p. A, Schematic diagram of conserved target sites of miR‐29b‐2‐5p in the 3'UTR of MEKK2 mRNA and miR‐34c‐3p in the 3'‐UTR of MEKK2 mRNA and NOTCH2 mRNA. B, Western blot detected levels of E‐cadherin, Vimentin, Fibronectin and α‐SMA in A549 and BEAS‐2B cells for the indicated groups. C, Luciferase reporter assays of relative luciferase activity of in A549 transfected with MEKK2‐wt or MEKK2‐mut (mean ± SD, n = 3), with **P < .01 vs the NC‐mimic group. D, Luciferase reporter assays of relative luciferase activity of in A549 transfected with MEKK2‐wt/MEKK2‐mut or NOTCH2‐wt /NOTCH2‐mut (mean ± SD, n = 3), with **P < .01 and ***P < .01 vs the NC‐mimic group. E, qRT‐PCR analysis of MEKK2 and NOTCH2 mRNA expression in A549 cells transfected with MEKK2 siRNA, NOTCH2 siRNA or NC‐siRNA (mean ± SD, n = 3), **P < .01. F, Western blot and densitometric analysis of MEKK2, E‐cadherin, Vimentin, Fibronectin, and α‐SMA in A549 cells transfected with MEKK2 siRNA or its negative control then treated with 5 ng/mL TGF‐β1 for 48 h (mean ± SD, n = 3), *P < .05. G, Western blot and densitometric analysis of NOTCH2, E‐cadherin, Vimentin, Fibronectin and α‐SMA in A549 cells transfected with NOTCH2 siRNA or its negative control then treated with 5 ng/mL TGF‐β1 for 48 h (mean ± SD, n = 3), *P < .05
FIGURE 5
FIGURE 5
MiR‐29b‐2‐5p or miR‐34c‐3p accelerates silica‐induced pulmonary fibrosis resolution by regulating EMT. A, Histological changes and collagen deposition in lung tissues were measured by haematoxylin and eosin (H&E) staining, Masson trichrome staining and IHC staining of collagen I. B, Hydroxyproline content of the lung tissues was used to assess the degree of collagen deposition. C, Western blot analysis of the protein expression of E‐cadherin, Vimentin, Fibronectin and α‐SMA in mouse lung tissues. D, qRT‐PCR analysis of miR‐29b‐2‐5p or miR‐34c‐3p expression in mouse fibrotic lung tissue on days 7, 14 and 28, U6 was used as an internal control, with *P < .05 vs the saline group. E, qRT‐PCR analysis of miR‐29b‐2‐5p and miR‐34c‐3p expression in mouse fibrotic lung tissue on control, SiO2, SiO2 + NC‐agomir, SiO2 + miR‐29b‐2‐5p agomir, and SiO2 + miR‐34c‐3p agomir groups, *P < .05. F, The histology of the lung lesions was observed with haematoxylin and eosin (H&E) staining, Masson trichrome staining and IHC staining of collagen I. G, Hydroxyproline content of the lung tissues was used to assess the degree of collagen deposition. H, The protein expression of E‐cadherin, Vimentin, Fibronectin and α‐SMA in mouse lung tissues treated with miR‐29b‐2‐5p or miR‐34c‐3p agomir for 28 d were determined by Western blot
FIGURE 6
FIGURE 6
Combination of miR‐29b‐2‐5p and miR‐34c‐3p exerts a synergic effect on EMT and silica‐induced pulmonary fibrosis. A, Western blot detected levels of E‐cadherin, Vimentin, Fibronectin and α‐SMA in A549 and BEAS‐2B cells for the indicated groups. B, qRT‐PCR analysis of miR‐29b‐2‐5p and miR‐34c‐3p expression in mouse fibrotic lung tissue on saline, SiO2, SiO2 + AAV‐control, SiO2 + AAV‐miR‐29b‐2‐5p, SiO2 + AAV‐miR‐34c‐3p and SiO2 + AAV‐miR‐mix, with *P < .05 vs the saline group. C, The histology of the lung lesions was observed with haematoxylin and eosin (H&E) staining, Masson trichrome staining and IHC staining of collagen I. D, Hydroxyproline content of the lung tissues was used to assess the degree of collagen deposition. E, Western blot and densitometric analysis of E‐cadherin, Vimentin, Fibronectin and α‐SMA in mouse lung tissues treated with saline, SiO2, SiO2 + AAV‐control, SiO2 + AAV‐miR‐29b‐2‐5p, SiO2 + AAV‐miR‐34c‐3p and SiO2 + AAV‐miR‐mix (mean ± SD, n = 3)
FIGURE 7
FIGURE 7
Schematic illustrations explain the signalling mechanisms by which lncRNA‐ATB regulates TGF‐β1‐induced EMT. LncRNA‐ATB acts as a sponge for miR‐29b‐2‐5p and miR‐34c‐3p, thereby promoting EMT and silica‐induced pulmonary fibrosis by regulating MEKK2 and NOTCH2
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