miR-29b Regulates TGF-β1-Induced Epithelial-Mesenchymal Transition by Inhibiting Heat Shock Protein 47 Expression in Airway Epithelial Cells

Abstract

Tissue remodeling contributes to ongoing inflammation and refractoriness of chronic rhinosinusitis (CRS). During this process, epithelial-mesenchymal transition (EMT) plays an important role in dysregulated remodeling and both microRNA (miR)-29b and heat shock protein 47 (HSP47) may be engaged in the pathophysiology of CRS. This study aimed to determine the role of miR-29b and HSP47 in modulating transforming growth factor (TGF)-β1-induced EMT and migration in airway epithelial cells. Expression levels of miR-29b, HSP47, E-cadherin, α-smooth muscle actin (α-SMA), vimentin and fibronectin were assessed through real-time PCR, Western blotting, and immunofluorescence staining. Small interfering RNA (siRNA) targeted against miR-29b and HSP47 were transfected to regulate the expression of EMT-related markers. Cell migration was evaluated with wound scratch and transwell migration assay. miR-29b mimic significantly inhibited the expression of HSP47 and TGF-β1-induced EMT-related markers in A549 cells. However, the miR-29b inhibitor more greatly induced the expression of them. HSP47 knockout suppressed TGF-β1-induced EMT marker levels. Functional studies indicated that TGF-β1-induced EMT was regulated by miR-29b and HSP47 in A549 cells. These findings were further verified in primary nasal epithelial cells. miR-29b modulated TGF-β1-induced EMT-related markers and migration via HSP47 expression modulation in A549 and primary nasal epithelial cells. These results suggested the importance of miR-29b and HSP47 in pathologic tissue remodeling progression in CRS.

Keywords: epithelial–mesenchymal transition; heat shock protein 47; microRNA; primary nasal epithelial cells; tissue remodeling; transforming growth factor beta-1.

Figure 1

TGF-β1 reduced miR-29b and induced HSP47 expression in A549 cells. (a) The putative binding sites of miR-29b and HSP47 were predicted using TargetScan (www.targetscan.org). A549 cells were treated with TGF-β1 at the indicated doses (0.5, 1, 2.5, 5 or 10 ng/mL, 24 h). (b) miR-29b and (c) HSP47 mRNA expression were measured using qPCR. A549 cells were treated with TGF-β1 (1 ng/mL) at the indicated dose (0.5, 1, 2.5, 5 or 10 ng/mL, 72 h). (d) HSP47 protein expression was measured using Western blotting. Data are expressed as the mean ± SEM of three independent experiments. * p < 0.05 vs. control, ** p < 0.001 vs. control.

Figure 2

Overexpression of miR-29b inhibited mRNA and protein expression levels of TGF-β1-induced EMT markers in A549 cells. A549 cells were stimulated with TGF-β1 (5 ng/mL) with miR control or miR-29b mimic. (a,b) mRNA expression levels of miR-29b and HSP47 were determined using qPCR. (c) HSP47 luciferase activity was measured by luciferase assay. (d) E-cadherin, α-SMA, vimentin, and fibronectin mRNA levels were analyzed through qPCR. (e) Protein expression levels of HSP47, E-cadherin, α-SMA, vimentin, and fibronectin were determined using Western blotting. (f) The cells were treated with TGF-β1 for 72 h after transfection of miR-29b mimic and then assessed for HSP47 (1st line, green), vimentin (1st line, red), α-SMA (2nd, green), and E-cadherin (2nd, red) expression/localization using immunofluorescence. Nuclei were stained with DAPI (blue). Scale bar = 20 μm. Values are expressed as mean ± SEM of three independent samples. * p < 0.05, vs. control + miR Control; † p < 0.05, vs. TGF-β1 + miR Control.

Figure 3

Inhibition of miR-29b expression induced mRNA and protein expression levels of TGF-β1-induced EMT markers in A549 cells. A549 cells were stimulated with TGF-β1 (5 ng/mL) with miR control or a miR-29b inhibitor. (a,b) The mRNA expression levels of miR-29b and HSP47 were analyzed using qPCR. (c) HSP47 luciferase activity was measured by luciferase assay. (d) The mRNA levels of EMT-related markers were measured using qPCR. (e) Protein expression levels of HSP47, E-cadherin, α-SMA, vimentin and fibronectin were determined using Western blotting. (f) The cells were treated with TGF-β1 for 72 h after transfection with miR-29b inhibitor, and then assessed for HSP47 (1st line, green), vimentin (1st line, red), α-SMA (2nd, green), and E-cadherin (2nd, red) expression/localization using immunofluorescence. Nuclei were stained with DAPI (blue). Scale bar = 20 μm. Values are expressed as mean ± SEM of three independent samples. * p < 0.05 vs. control + miR Control; † p < 0.05, vs. TGF-β1 + miR Control.

Figure 4

Inhibition of HSP47 induced mRNA and protein expression levels of TGF-β1-induced EMT markers in A549 cells. Specific HSP47 siRNAs were transfected prior to treatment with or without TGF-β1 for 24 h in A549 cells. (a,b) The mRNA expression levels of miR-29b and HSP47 were analyzed using qPCR. (c) The mRNA levels of EMT-related markers were determined using qPCR. (d) Specific HSP47 siRNAs were transfected prior to treatment with or without TGF-β1 for 72 h. Protein levels of HSP47 and EMT-related markers were evaluated using Western blotting. (e) Confocal laser scanning microscope was used to detect immunofluorescence. Representative fluorescein immunocytochemical staining shows HSP47 (1st line, green), vimentin (1st line, red), α-SMA (2nd line, green), and E-cadherin (2nd line red) with nuclear DAPI (blue). Scale bar = 20 μm. Data are expressed as the mean ± SEM of three independent experiments. * p < 0.05, vs. control + siControl; † p < 0.05, vs. TGF-β1 + siControl.

Figure 5

Overexpression of miR-29b contributed to A549 cell migration. A549 cells were seeded and transfected with miR control or miR-29b mimic before treatment with TGF-β1 for 72 h. (a) A wound scratch migration assay was performed to investigate the migration of A549 cells. Microscopic observation was performed 72 h after scratching the surface of a confluent layer of cells. (b) Inhibitory effects of the miR-29b mimic are also shown using a transwell migration assay. Scale bar = 100 μm. Data are expressed as the mean ± SEM of three independent experiments. * p < 0.05, vs. control + miR Control; † p < 0.05, vs. TGF-β1 + miR Control.

Figure 6

Silencing of HSP47 expression contributed to A549 cell migration. A549 cells were seeded and transfected with siControl or siHSP47 before treatment with TGF-β1 for 72 h. (a) A wound scratch migration assay was performed to investigate the migration of A549 cells. Microscopic observation was performed 72 h after scratching the surface of a confluent layer of cells. (b) Inhibitory effects of siHSP47 are shown using a transwell migration assay. Scale bar = 100 μm. Data are expressed as the mean ± SEM of three independent experiments. * p < 0.05, vs. control + siControl; † p < 0.05, vs. TGF-β1 + siControl.

Figure 7

Overexpression of miR-29b and silencing of HSP47 inhibited EMT induced by TGF-β1 in primary nasal epithelial cells. Primary nasal epithelial cells were cultured and pretreated with or without miR-29b mimic and siHSP47 before stimulation with TGF-β1 for 72 h. (a,b) Effect of miR-29b mimic on HSP47, E-cadherin, α-SMA, vimentin, and fibronectin protein expression in TGF-β1-stimulated primary nasal epithelial cells, as determined by Western blotting and immunofluorescence. (c,d) Effect of siHSP47 on HSP47, E-cadherin, α-SMA, vimentin, and fibronectin protein expression in TGF-β1-stimulated primary nasal epithelial cells determined by western blotting and immunofluorescence. Representative fluorescein immunocytochemical staining shows E-cadherin (red) and vimentin (green) with nuclear DAPI (blue). Scale bar = 20 μm. Data are expressed as the mean ± SEM of three independent experiments.