| Objective:Arsenic is known as a well-established human carcinogen.However,the mechanism of arsenic carcinogenesis remains incompletely understood.Epithelial mesenchymal transition(EMT)is characterized as a crucial morphological event in cancer progress.In this study,our purpose is to investigate the mechanisms of autophagy on chronic sodium arsenite-induced EMT in human bronchial epithelial cells,whereby to highlight a potential novel pathway in prevention and therapy of arsenite-exposed related illnesses.Methods:1.Cell treatment.For the acute exposure experiments,human bronchial epithelial BEAS-2B cells were treated with 2.5 μM sodium arsenite for 48 h;for chronic exposure,cells were maintained in 0.25 μM sodium arsenite for 72 h per passage,around 16 weeks.The transformed cells of BEAS-2B,designated here as TB2B,were the chronic exposed cells that were derived from colonies grown on soft agar.2.Establishment of stably BEAS-2B/GFP-LC3 cell lines.BEAS-2B cells were transfected by plasmid GFP-LC3 with Lipofectamine 2000 and selected with G418 after48 h for 12 days,following manufactures’ s standard protocols,and corroborated by western blot analysis.3.Detection of autophagy in chronic arsenic-induced BEAS-2B cells.The numbers of autophagosomes were observed by fluorescence microscopy in BEAS-2B/GFP-LC3 cells cultured in 2.5 μM sodium arsenite for 48 h.The expression of the autophagy proteins(Beclin-1,p62 and LC3-Ⅱ)in cells treated with chronic sodium arsenite were assessed by immunoblotting.4.Effect of chronic sodium arsenite on EMT and MEK/ERK1/2 signaling pathway.The expression of ERK1/2,p-ERK1/2,E-cadherin and Vimentin was confirmed by immunoblotting in TB2 B cells transfected with ERK1/2 siRNA.The relative expression levels of mRNA for the ZEB1,Snail and Twist genes,all of which are known EMT-related transcription factors,were measured based on qRT-PCR analysis,and their level of proteins were performed by western blot analysis in TB2 B cells.5.Effect of autophagy inhibition on the EMT and MEK/ERK1/2 signaling pathway.We performed soft agar assays and wound healing assay to assess colony formation capability and migration capability in TB2 B cells with autophagy inhibitor baf A1(10n M).Western blot was conducted to measure the levels of EMT and MEK/ERK1/2signaling pathways.6.Effect of autophagy gene knockout on the EMT.CRISPR/Cas9 system was designed to edit TB2 B cells and thusly developed a TB2 B beclin-1 knockout(KO)cell line.The levels of EMT markers in KO cells were detected by immunoblotting.The relative migration capacity was evaluated by transwell assay.7.Effect of MEK/ERK1/2 blockade on autophagy in TB2 B cells.The expression of Beclin-1,p-ERK1/2,Vimentin and Snail were measured by immunoblotting in TB2 B cells treated with MEK/ERK1/2 inhibitor U0126.8.Statistical analysis.All experiments were performed at three times independently.Differences in data were determined with one-way analysis of variance(ANOVA).P<0.05 was considered statistically significant.Results:1.The numbers of autophagosomes were upregulated by sodium arsenite in BEAS-2B cells.An increased number of autophagosomes labeled by GFP-LC3 puncta was observed in cells cultured in 2.5 μM sodium arsenite-containing media for 48 h(P<0.05).Furthermore,increased conversion of LC3-I isoform into LC3-Ⅱ isoform(P<0.01),overexpression of Beclin-1(P<0.01)and decreased expression of p62 were observed(P<0.05).2.Autophagy was activated by chronic sodium arsenite.Beclin-1 and conversion of LC3-Ⅰ isoform into the LC3-Ⅱ isoform were expressed at high levels(P<0.01,P<0.001),inversely,p62 at low level in BEAS-2B cells induced by chronic sodium arsenite(P<0.001).3.EMT was induced by chronic sodium arsenite exposure.The migratory capacity and the number and size of colonies were greater(P<0.05,P<0.01),the expression of E-cadherin was decreased(P<0.001),concomitantly vimentin was increased(P<0.001)in BEAS-2B cells induced by chronic sodium arsenite exposure.4.Chronic sodium arsenite exposure activates EMT via MEK/ERK1/2 signaling pathway.Compared with BEAS-2B cells,the expression of p-ERK1/2 was increased in TB2 B cells(P<0.001).ERK1/2 siRNA dampens Vimentin expression and inhibits cell migration.Western blot analysis confirmed that the expression of ZEB1 and Snail are significantly enhanced(P<0.001,P<0.01),however,Twist protein is suppressed(P<0.01)in TB2 B cells compared to BEAS-2B cells.Remarkably,quantitative reverse-transcriptase PCR confirmed increasing ZEB1 mRNA and Snail mRNA(P<0.05,P<0.01),nevertheless the Twist m RNA was not a significant difference in TB2 B cells compared with BEAS-2B cells(P>0.05).5.Turning off autophagy promotes EMT,as well as the MEK/ERK1/2 signaling pathway further activation.Inhibition of autophagy with autophagy inhibitor baf A1(10nM)results in high migration and colony formation capability(P<0.01,P<0.01).Impressively,the phosphorylation of ERK1/2(P<0.05)and the expression of Snail(P<0.001)and ZEB1(P<0.01)with simultaneous loss of E-cadherin(P<0.05)were further increased.6.Autophagy gene knockout potentiates EMT activation.TB2 B beclin-1 KO cells had significantly increased accumulation of the mesenchymal protein Vimentin,ZEB1 and Snail(P<0.05,P<0.01,P<0.01)with concurrent reduction of E-cadherin(P>0.05).In addition,we found KO cells had more relative of cell migration compared to WT cells(P<0.01).7.The expression of Beclin-1 was not marginally affected by MEK/ERK1/2signaling pathway impairment.The strong decreased levels of p-ERK1/2(P<0.01),Snail(P<0.05)and Vimentin(P<0.01),but hardly decrease levels of Beclin-1(P>0.05)were observed in the presence of MEK/ERK1/2 inhibitor U0126.Conclusions:1.Autophagy was activated by chronic sodium arsenite exposure in BEAS-2B cells.2.EMT was induced by chronic sodium arsenite exposure via MEK/ERK1/2signaling pathway.3.Autophagy disor der promotes chronic sodium arsenite-induced EMT.4.Autophagy inhibits EMT induced by chronic sodium arsenite exposure via MEK/ERK1/2 signaling pathway. |
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