Korean J Physiol Pharmacol.  2022 Jul;26(4):239-253. 10.4196/kjpp.2022.26.4.239.

MicroRNA-21 promotes epithelial-mesenchymal transition and migration of human bronchial epithelial cells by targeting poly (ADP-ribose) polymerase-1 and activating PI3K/AKT signaling

• Zhang S
• Sun P
• Xiao X
• Hu Y
• Qian Y
• Zhang Q

• KMID: 2530943
• DOI: http://doi.org/10.4196/kjpp.2022.26.4.239

Abstract

Epithelial-mesenchymal transition (EMT ) is known to be involved in airway remodeling and fibrosis of bronchial asthma. However, the molecular mechanisms leading to EMT have yet to be fully clarified. The current study was designed to reveal the potential mechanism of microRNA-21 (miR-21) and poly (ADP-ribose) polymerase-1 (PARP-1) affecting EMT through the PI3K/AKT signaling pathway. Human bronchial epithelial cells (16HBE cells) were transfected with miR-21 mimics/inhibitors and PARP-1 plasmid/small interfering RNA (siRNA). A dual luciferase reporter assay and biotin-labeled RNA pull-down experiments were conducted to verify the targeting relationship between miR-21 mimics and PARP-1. The migration ability of 16HBE cells was evaluated by Transwell assay. Quantitative real-time polymerase chain reaction and Western blotting experiments were applied to determine the expression of Snail, ZEB1, E-cadherin, N-cadherin, Vimentin, and PARP-1. The effects of the PI3K inhibitor LY294002 on the migration of 16HBE cells and EMT were investigated. Overexpression of miR-21 mimics induced migration and EMT of 16HBE cells, which was significantly inhibited by overexpression of PARP-1. Our findings showed that PARP-1 was a direct target of miR-21, and that miR-21 targeted PARP-1 to promote migration and EMT of 16HBE cells through the PI3K/AKT signaling pathway. Using LY294002 to block PI3K/AKT signaling pathway resulted in a significant reduction in the migration and EMT of 16HBE cells. These results suggest that miR-21 promotes EMT and migration of HBE cells by targeting PARP-1. Additionally, the PI3K/AKT signaling pathway might be involved in this mechanism, which could indicate its usefulness as a therapeutic target for asthma.

Keyword

Asthma; Epithelial-mesenchymal transition; MicroRNAs; PI3K/AKT signaling pathway; Poly (ADP-ribose) polymerase-1

Figure

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  Fig. 1 PARP-1 inhibits the migration of 16HBE cells. (A, B) Western blot and qRT-PCR were performed to evaluate the protein and mRNA expressions of PARP-1 in 16HBE cells transfected with NC, PARP-1 plasmid, or PARP-1 siRNA after 48 h. The experiments were replicated three times and presented as mean ± SD. *p < 0.05 vs. NC group. (C) Effects of PARP-1 on migration in 16HBE cells were detected by Transwell assay. Scale bars = 50 μm. The experiments were replicated three times and presented as mean ± SD. PARP-1, poly (ADP-ribose) polymerase-1; 16HBE, Human bronchial epithelial cell; NC, normal control; siRNA, small interfering RNA; siPARP-1, PARP-1 siRNA. *p < 0.05 vs. NC group, **p < 0.01 vs. NC group.

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  Fig. 2 MiR-21 suppresses PARP-1 expression and PARP-1 is a direct target of miR-21. (A–C) The 16HBE cells were transfected with NC, miR-21 mimics, miR-21 inhibitor, miR-21 mimics + PARP-1 plasmid, or miR-21 inhibitor + PARP-1 siRNA. Expressions of miR-21 and PARP-1 mRNA were detected by qRT-PCR and the protein expression of PARP-1 was detected by Western blot 48 h post-transfection. The experiments were replicated three times and presented as mean ± SD. *p < 0.05 vs. NC group, **p < 0.01 vs. NC group, †p < 0.05 vs. miR21 or miR-21 inhibitor group, ††p < 0.01 vs. miR-21 inhibitor group. (D) The potential binding site between miR-21 and 3’UTR of PARP-1 mRNA is shown. Luciferase activities of 16HBE cells transfected with NC, miR-21 mimics, PARP-1 Wt, PARP-1 Mut, miR-21 mimics + PARP-1 Wt, or miR-21 mimics + PARP-1 Mut were measured by luciferase reporter assay. The experiments were replicated three times and presented as mean ± SD. **p < 0.01 vs. NC group. (E) Western blot was used to detect the binding capacity of biotin-labeled miR-21 mimics probes with PARP-1 protein. The experiments were replicated three times. miR-21, microRNA-21; PARP-1, poly (ADP-ribose) polymerase-1; 16HBE, Human bronchial epithelial cell; NC, normal control; siRNA, small interfering RNA; siPARP-1, PARP-1 siRNA; UTR, untranslated region; Wt, wild type; Mut, mutant.

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  Fig. 3 Effects of miR-21/PARP-1 on migration of 16HBE cells. (A, B) The 16HBE cells were transfected with NC, miR-21 mimics, miR-21 inhibitor, miR-21 mimics + PARP-1 plasmid, or miR-21 inhibitor + PARP-1 siRNA. Transwell assay was performed to assess the effects of miR-21/PARP-1 on the migration of 16HBE cells. Scale bars = 50 μm. The experiments were replicated three times and presented as mean ± SD. ***p < 0.001 vs. NC group, †††p < 0.001 vs. miR21 inhibitor group, ††††p < 0.0001 vs. miR21 group. (C) Images of the cell morphology 72 h post transfection. Scale bars = 50 μm. miR-21, microRNA-21; PARP-1, poly (ADP-ribose) polymerase-1; 16HBE, Human bronchial epithelial cell; NC, normal control; siRNA, small interfering RNA; siPARP-1, PARP-1 siRNA.

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  Fig. 4 Effects of PARP-1 on expression of EMT-related proteins and EMT-regulating transcription factors. (A, C) Western blot analysis evaluated the expression of Snail, ZEB1, N-cadherin, Vimentin and E-cadherin in 16HBE cells transfected with NC, PARP-1 plasmid, or PARP-1 siRNA after 48 h. The experiments were replicated three times and presented as mean ± SD. *p < 0.05 vs. NC group, **p < 0.01 vs. NC group. (B, D) The mRNA expressions of Snail, ZEB1, N-cadherin, Vimentin, and E-cadherin in 16HBE cells transfected with NC, PARP-1 plasmid, or PARP-1 siRNA after 48 h, as detected by qRT-PCR. The experiments were replicated three times and presented as mean ± SD. PARP-1, poly (ADP-ribose) polymerase-1; EMT, epithelial-mesenchymal transition; 16HBE, Human bronchial epithelial cell; NC, normal control; siRNA, small interfering RNA; siPARP-1, PARP-1 siRNA. *p < 0.05 vs. NC group, **p < 0.01 vs. NC group, ***p < 0.001 vs. NC group.

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  Fig. 5 Overexpression of PARP-1 alleviates the EMT induced by miR-21 mimics in 16HBE cells. (A, C, E, G) Western blot analysis evaluated the protein expression of Snail, ZEB1, N-cadherin, Vimentin, and E-cadherin in 16HBE cells transfected with NC, miR-21 mimics, miR-21 inhibitor, miR-21 mimics + PARP-1 plasmid or miR-21 inhibitor + PARP-1 siRNA after 48 h. The experiments were replicated three times and presented as mean ± SD. *p < 0.05 vs. NC group, **p < 0.01 vs. NC group, †p < 0.05 vs. miR21 or miR21 inhibitor group, ††p < 0.01, vs. miR21 or miR21 inhibitor group. (B, D, F, H) The mRNA expression of Snail, ZEB1, E-cadherin, Vimentin, and E-cadherin in 16HBE cells transfected with NC, miR-21 mimics, miR-21 inhibitor, miR-21 mimics + PARP-1 plasmid, or miR-21 inhibitor + PARP-1 siRNA after 48 h were detected by qRT-PCR. The experiments were replicated three times and presented as mean ± SD. PARP-1, poly (ADP-ribose) polymerase-1; EMT, epithelial-mesenchymal transition; miR-21, microRNA-21; 16HBE, Human bronchial epithelial cell; NC, normal control; siRNA, small interfering RNA; siPARP-1, PARP-1 siRNA. *p < 0.05 vs. NC group, **p < 0.01 vs. NC group, ***p < 0.001 vs. NC group, ****p < 0.0001 vs. NC group, †p < 0.05 vs. miR21 inhibitor group, ††p < 0.01 vs. miR21 group, †††p < 0.001 vs. miR21 group.

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  Fig. 6 Effects of miR-21/PARP-1 on the PI3K/AKT signaling pathway. (A–C) Western blot analysis evaluated the expression of PI3K/AKT-signaling-pathway-related molecular proteins in 16HBE cells transfected with NC, miR-21 mimics, miR-21 inhibitor, PARP-1 plasmid, PARP-1 siRNA, miR-21 mimics + PARP-1 plasmid, or miR-21 inhibitor + PARP-1 siRNA after 48 h. The experiments were replicated three times and presented as mean ± SD. PARP-1, poly (ADP-ribose) polymerase-1; 16HBE, Human bronchial epithelial cell; NC, normal control; miR-21, microRNA-21; siRNA, small interfering RNA; siPARP-1, PARP-1 siRNA. *p < 0.05 vs. NC group, **p < 0.01 vs. NC group, ***p < 0.001 vs. NC group, †p < 0.05 vs. miR21 or miR-21 inhibitor group, ††p < 0.01 vs. miR21 inhibitor group.

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  Fig. 7 PI3K inhibitor LY294002 inhibits the migration of 16HBE cells. (A, B) The PI3K inhibitor LY294002 was used to study effects on the migration of 16HBE cells transfected with NC, miR-21 mimics, or PARP-1 siRNA. The experiments were replicated three times and presented as mean ± SD. 16HBE, Human bronchial epithelial cell; NC, normal control; miR-21, microRNA-21; PARP-1, poly (ADP-ribose) polymerase-1; siRNA, small interfering RNA; siPARP-1, PARP-1 siRNA; D, dimethylsulfoxide; LY, LY294002. Scale bars = 50 μm. *p < 0.05 vs. NC + D group, ***p < 0.001 vs. NC + D group, ††p < 0.01 vs. siPARP-1 + D group, †††p < 0.001 vs. miR21 + D group.

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  Fig. 8 MiR-21/PARP-1 regulates the EMT of 16HBE cells via activation of the PI3K/AKT signaling pathway. (A, B) Western blot analysis evaluated the protein expressions of PI3K/AKT-signaling-pathway-related molecular proteins in 16HBE cells transfected with NC, miR-21 mimics, or PARP-1 siRNA after 48 h. The experiments were replicated three times and presented as mean ± SD. *p < 0.05 vs. NC + D group, **p < 0.001 vs. NC + D group, †p < 0.05 vs. miR21 + D or siPARP-1 + D group. (C–F) Effects of LY294002 on the EMT-related proteins in 16HBE cells transfected with NC, miR-21 mimics, or PARP-1 siRNA, as detected by Western blot and qRT-PCR after 48 h. The experiments were replicated three times and presented as mean ± SD. miR-21, microRNA-21; PARP-1, poly (ADP-ribose) polymerase-1; EMT, epithelial-mesenchymal transition; 16HBE, Human bronchial epithelial cell; NC, normal control; siRNA, small interfering RNA; siPARP-1, PARP-1 siRNA; D, dimethylsulfoxide; ns, not significant; LY, LY294002. *p < 0.05 vs. NC + D group, **p < 0.01 vs. NC + D group, ***p < 0.001 vs. NC + D group, ****p < 0.0001 vs. NC + D group, †p < 0.05 vs. miR21 + D or siPARP-1 + D group, ††p < 0.01 vs. miR21 + D or siPARP-1 + D group, †††p < 0.001 vs. miR21 + D or siPARP-1 + D group.

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  Fig. 9 Schematic diagram illustrating how miR-21/PARP-1 activates the PI3K/AKT signaling pathway to induce EMT and migration of 16HBE cells. miR-21, microRNA-21; PARP-1, poly (ADP-ribose) polymerase-1; EMT, epithelial-mesenchymal transition; 16HBE, Human bronchial epithelial cell.

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