Upregulation Of Hypoxia-inducible Factor-1α Contributes To Hexavalent Chromium-induced Expression Of Mesenchymal And Stem Cell Markers In Human Bronchial Epithelial Cells

Background Hexavalent chromium(Cr(Ⅵ))is a common environmental and occupational carcinogen,which can cause a series of health damage such as lung cancer.Epithelial mesenchymal transition plays a key role in the development of various lung diseases,which is closely related cancer stem cells.However,it is unclear whether hexavalent chromium can lead to EMT and cancer stem cells-like characteristics of lung cells.Hypoxia may play a key role in the formation and development of cancer stem cells,but the molecular mechanism of Cr(Ⅵ)inducing cell hypoxia then leading to the formation of cancer stem cells has not been fully elucidated.Objective The pulmonary bronchial epithelial cell line(BEAS-2B)was used as a model.We explore the potential mechanism of HIF-1α on Cr(Ⅵ)inducing epithelial mesenchymal transformation(EMT)and the formation of cancer stem cells(CSCs)through in vitro models,which is of great significance to induce EMT and the formation of CSCs.Methods BEAS-2B cells in good were used for following experiments:1.To verify the expression of HIF-1α in BEAS-2B cells exposed to Cr(Ⅵ),MTT assay was used to define the concentration of Cr(Ⅵ)in BEAS-2B cells.Western Blot assay was used to determine the expression level of HIF-1α protein in cells after Cr(Ⅵ)exposure.q RT-PCR assay was used to determine HIF-1α m RNA expression in cells after Cr(Ⅵ)exposure.2.To study the process of HIF-1α on Cr(Ⅵ)induced EMT and CSCs,phase contrast microscope was used to observe the morphology of Cr(Ⅵ)cells exposed for a long time.The migratory ability of chronically exposed Cr(Ⅵ)cells was observed by the scratch assay;the ability for adherent cell proliferation was determined by the colony formation assay;the ability for self-renewal of cells was determined by the sphere formation assay;the levels of HIF-1α,EMT-related proteins and stem cell marker proteins before and after HIF-1α inhibition were determined by Western Blot analysis.3 To discuss the mechanism of HIF-1α on Cr(Ⅵ)induced EMT and cancer stem cell,Western Blot was used to detect the protein expression level of TGF-β/Smads,MAPKs and Wnt/β-catenin signaling pathway related protein before and after using HIF-1α inhibition.Cellular immunofluorescence assay was used to observe β-catenin protein distribution.Results1.After Cr(Ⅵ)with different concentrations(24 h-0,0.125,0.25,0.5,1.0 μM)and different times(0.25 μM-0,6,12,24,36 h)in BEAS-2B cells,the protein and m RNA expression of HIF-1α were up-regulated.2.Long-term exposure to Cr(Ⅵ)induced changes in cell morphology and enhanced cell migration.The expression level of E-cadherin was down regulated,while vimentin was up regulated with an obvious time-does effect relationship.3.HIF-1α inhibitor YC-1 protects BEAS-2B cells from Cr(Ⅵ)inducing EMT.4.Long-term exposure to Cr(Ⅵ)enhances the self-renewal ability and long-term growth ability of BEAS-2B cells.The expression of cancer stem cell marker proteins in cells were up-regulated in a time-does effect relationship.5.HIF-1α inhibitor YC-1 protects BEAS-2B cells from Cr(Ⅵ)acquiring cancer stem cell like characteristics.After YC-1,the expression of CSCs related proteins were decreased.6.Cr(Ⅵ)actives TGF-β1/SMAD2/3 and MAPKs signal pathway in lung epithelial cells.BEAS-2B cells were treated with Cr(Ⅵ)of different concentrations and dosages for 24 h and exposed to 0.25 μM/L at different times,EMT classical signaling pathway(TGF-β1/Smad2/3)related proteins and non-classical signal pathways(MAPKs)related proteins were up-regulated.7.HIF-1α inhibitor YC-1 protects the activation of TGF-β1/Smad2/3 and MAPKs pathway.After YC-1,the expression of TGF-β1/Smad2/3 and MAPKs pathway related proteins were decreased.8.Cr(Ⅵ)actives Wnt/β-Catenin pathway.After Cr(Ⅵ)exposure,the expression of Wnt3 a and Cyclin D1 protein in cells were up-regulated in a time-effect relationship.9.HIF-1α inhibitor YC-1 protects the activation of Wnt/β-catenin pathway.Conclusions1.Cr(Ⅵ)can up-regulate HIF-1α expression in BEAS-2B cells.2.Cr(Ⅵ)may promote EMT and CSCs related proteins by up-regulate HIF-1αexpression in BEAS-2B cells.3.Cr(Ⅵ)may activate TGF-β/Smads,MAPKs and Wnt/β-catenin signaling pathway to promote EMT and CSCs by up-regulate HIF-1α expression in BEAS-2B cells.