| Background and objective: The irreversible pulmonary fibrosis induced by paraquat(PQ)poisoning is the main cause of death in patients.Epithelial-mesenchymal transition(EMT)plays an important role in the formation of pulmonary fibrosis.This study was designed to investigate the effect of hypoxia-inducible factor-1?(HIF-1?)and its target gene micro RNA-210(mi R-210)on EMT in PQ-induced pulmonary fibrosis,and the relationship between HIF-1? and mi R-210.Methods: The SD rats were randomly divided into control group and model group.The model group was administered with intragastric infusion of PQ at a dose of 50 mg/kg once.The rats were sacrificed at different time points(2,6,12,24,48 and 72 h).The executions were divided into six subgroups.In vitro,A549 and RLE-6TN cells were used to inhibit or overexpress HIF-1? and mi R-210,respectively,and treated with the corresponding final concentration of PQ for 24 h.Collected cells and rat lung tissue,western blot,q RT-PCR,co-immunoprecipitation and other techniques were used to detect changes.Results: After PQ poisoning in vitro and in vivo models,epithelial markers(ZO-1 and E-cadherin)expression decreased,and the interstitial marker(?-SMA)increased significantly,EMT occurred.At 2 h after PQ poisoning,the expression of HIF-1? in lung tissue of rats was significantly increased and was higher than that in the control group at 72 h.The silencing of HIF-1? in A549 and RLE-6TN cells significantly reduced the expression of epithelial markers,decreased the number of interstitial markers,significantly improved the morphological changes of cells,reduced the expression of Snail and ?-catenin,and attenuated the EMT induced by PQ.The mi R-210 in lungs of rats exposed to PQ for 6 h significantly increased,and mi R-210 decreased significantly after inhibiting HIF-1? in alveolar epithelial cells.After overexpression of mi R-210 in vitro,the expression of ?-SMA was further increased,the expressions of epithelial markers(ZO-1 and E-cadherin)were further decreased,the degree of EMT was increased,the expression of HIF-1? was significantly increased.After inhibiting mi R-210,significantly reduced PQ-induced EMT and HIF-1?.q RT-PCR results showed that there was no significant change in HIF-1? m RNA after mi R-210 was up-regulated or down-regulated.After overexpression of mi R-210,the expression of RUNX3 was significantly inhibited,the hydroxylation of HIF-1? was decreased,PHD2 bound to RUNX3 was also decreased,and the result was reversed after mi R-210 was inhibited.Luciferase assay confirmed that RUNX3 is one of the target genes of mi R-210.After RUNX3 was over-expressed in A549 cells,OH-HIF-1? increased and HIF-1? decreased.After inhibiting RUNX3,the results were reversed,and there was no significant change in the expression of PHD2.Conclusion: This study identified that EMT is involved in PQ poisoning-induced pulmonary fibrosis,and that HIF-1? may regulate EMT via the Snail and ?-catenin pathways.After PQ poisoning,mi R-210 was significantly increased,and was regulated by HIF-1?.mi R-210 can promote PQ-induced EMT.There is a positive feedback regulation between HIF-1? and mi R-210.Mi R-210 may reduce the hydroxyapatite-HIF-1? ability of PHD2 by inhibiting RUNX3,and increase the intracellular HIF-1?,regulating EMT on the one hand and further promoting mi R-210 expression,forming a vicious circle,continue to promote PQ-induced EMT and pulmonary fibrosis development.This study suggests that HIF-1? may be a potential target for PQ poisoning-induced pulmonary fibrosis. |
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