| Objective: To investigate the role and mechanism of IL-32β in the process of epithelial-mesenchymal transformation of human alveolar epithelial cells.Methods: Firstly,we used tunicamycin to treat A549 cells,and observed the morphology of A549 cells,detected the expression of IL-32 and its subtypes,and screened the most suitable subtype of IL-32.Secondly,Human lung adenocarcinoma A549 cells were treated with recombinant human(rh)IL-32β,and the influence on cell morphology was assessed microscopically,along with the expression of EMT-related markers using reverse transcription-quantitative polymerase chain reaction(RT-qPCR).Positive validation of the effect of IL-32β on A549 cells.To isolate the role of IL-32β in EMT,A549 cells were transfected with plasmids containing IL-32 small interfering(si)RNA and subsequently treated with the EMT inducer tunicamycin(TM).The EMT-related markers were detected by RT-qPCR and western blotting.Reverse confirmation of the role of IL-32β in EMT of A549 cells.Moreover,A549 cells were treated with 4-phenylbutyric acid(4-PBA),an ER stress inhibitor,and GRP78 expression was detected by RT-qPCR and western blotting.Results: 1.Expression of IL-32 and its subtypes was upregulated in A549 cells,IL-32β and IL-32γ were upregulated most significantly.2.rhIL-32β induced EMT in A549 cells: After treatment with 100 ng/ml rhIL-32β for 24 h,a portion of the A549 cells showed a morphological change from a pebble-like shape to an irregular elongated shape.Indeed,expression of the epithelial cell marker E-cadherin was significantly downregulated(P < 0.05),while the expression of the mesenchymal cell markers N-cadherin,vimentin,and Zeb-1 were significantly upregulated(P < 0.05)after rhIL-32β treatment.3.IL-32 siRNA inhibited TM-induced EMT: RT-qPCR showed that the mRNA expression levels of the mesenchymal cell markers N-cadherin,vimentin,Snail,and Zeb-1 were significantly increased in the TM group compared with those of the control group(P < 0.05),whereas these expression levels were significantly decreased in the TM+IL-32β siRNA group compared with those of the TM group(P < 0.05).4.rhIL-32β induced ER stress in A549 cells: Twenty-four hours after treatment of A549 cells with 100 ng/ml rhIL-32β,both the mRNA and protein expression levels of GRP78 increased significantly(P < 0.05).Moreover,treatment of rhIL-32β with 1.0 mM 4PBA significantly decreased the mRNA and protein expression levels of GRP78 compared with those of cells treated with rhIL-32β alone(P < 0.05).5.rhIL-32β promotes ER stress mediated EMT in A549 cells: To further ascertain the relationship between ER stress and EMT,the expression of EMT-related molecules was measured in A549 cells treated with the ER stress inhibitor 4-PBA.Compared with cells treated with rhIL-32β alone,4-PBA treatment reduced the mRNA expression levels of the mesenchymal cell markers N-cadherin,vimentin,and Zeb-1,and increased the mRNA expression level of the epithelial cell marker E-cadherin(P < 0.05).Similarly,the protein expression of N-cadherin and α-SMA were upregulated in the rhIL-32β group but significantly downregulated in the rhIL-32β+4PBA group(P < 0.05).6.IL-32β can induce inflammatory cytokine production in A549 cells: After rhIL-32β treatment,the mRNA expression levels of the inflammatory cytokines tumor necrosis factor(TNF)-α,IL-6,transforming growth factor(TGF)-β1,and IL-1β in the rhIL-32β group were significantly higher than those of the control group(P < 0.05).7.IL-32β up-regulates p-FAK expression in A549 cells: After rhIL-32β treatment,there was no difference in FAK protein expression between the control group and the IL-32β group(P > 0.05).But the protein expression levels of p-FAK in the rhIL-32β group were significantly higher than those of the control group(P < 0.05).Conclusion: Collectively,these results suggest that IL-32β induces EMT in A549 cells by triggering ER stress.These results suggest that IL-32 may be a novel marker for IPF. |
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