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. 2020 Oct 23;20(1):278.
doi: 10.1186/s12890-020-01319-z.

IL-32 induces epithelial-mesenchymal transition by triggering endoplasmic reticulum stress in A549 cells

Ling Gong  1   2 , Gang Liu  2   3 , Honglan Zhu  2 , Caihong Li  4 , Pengmei Li  4 , Changlu Liu  4 , Hongbo Tang  2 , Kaifeng Wu  2   5 , Jie Wu  2   5 , Daishun Liu  6   7 , Xiaoping Tang  8
Affiliations

IL-32 induces epithelial-mesenchymal transition by triggering endoplasmic reticulum stress in A549 cells

Ling Gong et al. BMC Pulm Med. .

Abstract

Background: Epithelial-mesenchymal transition (EMT) is a key process in the onset and development of idiopathic pulmonary fibrosis (IPF) with unclear mechanisms. Our previous studies found that bleomycin and tunicamycin could induce ER stress and consequently trigger EMT accompanying with IL-32 overexpression. This study was aimed to investigate the effects of IL-32 on EMT and ER stress to elucidate the pathogenesis of IPF.

Methods: Human lung adenocarcinoma A549 cells were treated with recombinant human (rh)IL-32, IL-32 siRNA and EMT inducer tunicamycin, or 4-phenylbutyric acid (4-PBA), respectively. Then the cell morphology was observed and the expression of ER-related markers and EMT-related markers were detected by RT-qPCR or western blotting.

Results: Stimulation of A549 cells with rhIL-32 led to a morphological change from a pebble-like shape to an elongated shape in a portion of the cells, accompanied by down regulated expression of the epithelial cell marker E-cadherin and up regulated expression of the mesenchymal cell markers N-cadherin, Vimentin, and Zeb-1. However, these rhIL-32 induced changes were inhibited by the ER stress inhibitor 4-PBA. Suppression of IL-32 expression with siRNA inhibited TM-induced EMT. Further stimulation of the A549 cells with rhIL-32 demonstrated an increase in the expression of GRP78, although this increase was also inhibited by 4-PBA.

Conclusions: These results suggest that IL-32 induces EMT in A549 cells by triggering ER stress, and IL-32 may be a novel marker for IPF.

Keywords: Endoplasmic reticulum stress; Epithelial-mesenchymal transition; IL-32; Idiopathic pulmonary fibrosis.

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Conflict of interest statement

None.

Figures

Fig. 1
Fig. 1
Morphology and IL-32 expression changes of TM treatment. a Morphologies of A549 cells of the control, solvent (DMSO)-treated, and TM-treated groups. b MTT assay of cell proliferation of control, solvent-treated, and TM-treated cells. c mRNA expression levels of IL-32, measured by RT-qPCR. * P < 0.05 compared with the control or solvent group
Fig. 2
Fig. 2
Morphology and EMT-related gene expressions in rhIL-32 treated cells. a Morphologies of A549 cells of the control and rhIL-32 treated groups observed under an inverted microscope. b mRNA expression levels of EMT-related molecular markers, measured by RT-qPCR. c, d Protein expression levels of EMT-related molecular markers measured by western blotting. *P < 0.05 compared with the control group
Fig. 3
Fig. 3
Inhibition of EMT in A549 cells by IL-32 siRNA. a Transfection efficiency of A549 cells: A549 were cultured in vitro and inoculated onto six-well plates. When the cells reach confluence, Lipofectamine 3000 transfection reagent was used to transfect A549 cells with fluorescent plasmids containing IL-32 siRNA. Forty-eight hours after transfection, the IL-32 shRNA transfection was evaluated by observing the intensity of green fluorescent protein with an inverted fluorescence microscope. The IL-32 gene silencing efficiency was measured using RT-qPCR b and western blotting assay c, d. e The mRNA expression levels of EMT-related molecular markers after transfection with IL-32 siRNA. *P < 0.05 compared with the control group, **P < 0.05 compared with the TM group. f, g The protein expression levels of EMT-related molecular markers after transfection with IL-32 siRNA. *P < 0.05 compared with the control group, **P < 0.05 compared with the TM group
Fig. 4
Fig. 4
GRP78 expression characteristics induced by IL-32 or 4-PBA. a GRP78 mRNA expression levels. *P < 0.05 compared with the control group, **P < 0.05 compared with the rhIL-32 group. b and c GRP78 protein expression levels, measured by western blotting. *P < 0.05 compared with the control group, **P < 0.05 compared with the rhIL-32 group
Fig. 5
Fig. 5
The effect of IL-32 or 4-PBA on the expression of EMT related genes and effect of IL-32 on the expression of inflammatory cytokine. a mRNA expression levels of EMT-related marker genes. * P < 0.05 compared with the control group, ** P < 0.05 compared with the rhIL-32 group. b and c Protein expression levels of EMT-related proteins measured by western blotting. *P < 0.05 compared with the control group, **P < 0.05 compared with the rhIL-32 group. d mRNA expression of inflammatory cytokines in control and rhIL-32 treated A549 cells. *P < 0.05 compared with the control group. e Protein expression of inflammatory cytokines in control and rhIL-32 treated A549 cells measured by ELISA assay. *P < 0.05 compared with the control group
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