The Role Of NFE2L1 In The Arsenic-induced Malignant Transformation Of Human Bronchial Epithelial Cells And Its Underlying Mechanism

Objective:Arsenic is a human carcinogen recognized by the International Agency for Research on Cancer?IARC?.The cancer induced by arsenic exposure in occupational or non-occupational environments has attracted much attention,but the mechanism of arsenic carcinogenesis is still eliusive.The lung is an important target organ for arsenic carcinogenesis.Epidemiological investigations suggest a close relationship between arsenic exposure and lung cancer.Epithelial mesenchymal transition?EMT?is a pathological process closely related to the development of tumors,which provides a molecular genetic basis for tumor cells to get rid of their epithelial characteristics and obtain migration characteristics.After undergoing such changes,the cells can then invade the tissue surrounding the primary tumor,exudate into the lymphatic vessels or blood vessels,and circulate into the distant place,eventually colonizing the metastatic niche.E-cadherin and SNAIL are important molecular markers of the EMT process.During the EMT process,the cells show a decrease in E-cadherin and an increase in its inhibitor SNAIL due to the loss of epithelial characteristics.NFE2L1 is important for regulating the oxidative homeostasis of the body and maintaining protein balance and metabolic homeostasis.The protective effect of its family member NFE2L2 in preventing chemical and radioactive?ionizing,ultraviolet?carcinogenesis has been well studied.However,NFE2L2 also confers chemo-chemotherapy resistance to malignant cells and promotes cancer development by mediating molecular events such as acquired anti-apoptosis and metabolic reprogramming.Because NFE2L1 and NFE2L2 have many overlapping functional structures,NFE2L1 may become an important new target for the prevention and treatment of arsenic induced lung cancer.In the present study,a human bronchial epithelial cell model chronically exposed to environmenrelavant doses of arsenic was constructed to investigate the potential role of NFE2L1 in EMT and malignant transformation.The study is aim to provide new clues for the prevention and treatment of arsenic-induced lung cancer.Methods:1.The chronic exposure of BEAS-2B human bronchial epithelial cells to inorganic arsenic:BEAS-2B cells were treated with 0.1?mol/L,0.2?mol/L,0.5?mol/L NaAsO₂?As??including NFE2L1-L gene silencing cells and control cells?.The control group was the same algebraic cells that were not treated with As.The cells were passaged after fusion to 90%and medium was replaced every other day.Cell samples were collected at12 weeks,24 weeks,30 weeks,and 36 weeks.2.Inorganic arsenic acute treatment of BEAS-2B cells:BEAS-2B cells were treated with 20?mol/L NaAsO₂ for 6 h,solvent control.Then cell samples were collected,and NFE2L1-L protein expression was detected by Western blot.3.NFE2L1-L gene silencing and identification:BEAS-2B cells were transfected with lentivirus carrying NFE2L1-L shRNA targeting?NFE2L1-L-KD?and non-target negative control lentivirus?SCR?,then were treated with 1?g/mL puromycin for screensing stably infected cells.The transcription level of NFE2L1-L was detected by RT-qPCR.Scramble and NFE2L1-L-KD cells were treated with 20?mol/L NaAsO₂ for 6 h,and protein expression levels of NFE2L1-L were detected by western blot.4.EMT assessment:BEAS-2B RNA and protein samples were received when cells exposed to chronic arsenic for 12 weeks,24 weeks,30 weeks,and 36 weeks,respectively,reverse Transcript Quantitative PCR?RT-qPCR?was used to detect the transcription of E-cadherin,SNAIL1 and TWIST2 at the mRNA level,and the protein level of E-cadherin and Vimentin was detected by western blot.The above indicators evaluated the process of cell epithelial mesenchymal transition?EMT?.5.Detection of indicators related to malignant transformation:BEAS-2B cells were treated with 0.1?mol/L,0.2?mol/L,0.5?mol/L NaAsO₂.The cells were collected at 12weeks,24 weeks,30 weeks and 36 weeks,and the cell proliferation ability was detected by colony formation assay.The cell migration ability was measured by cell scratch healing test.They were used to evaluate the degree of malignant transformation.SCR cells and NFE2L1-L-KD cells exposed to chronic arsenic at 12 weeks and 36 weeks were inoculated into 35 mm culture dishes.After 24 h,the cell morphology was taken under microscope.6.Overexpression and identification of NFE2L1-L gene:transfection of human renal epithelium with lentivirus?NFE2L1-L-OE?carrying NFE2L1-L shRNA targeting and non-target negative control lentivirus?Scramble?Cells?293T?were screened for stable infected cells with 5?g/mL blasticidin.The transcription level of NFE2L1-L was detected by RT-qPCR.293T?NFE2L1-L-OE/Scramble?cells were treated with 20nmol/L erythropoietin?EPO?for 6 h,and the expression level of NFE2L1-L protein was detected by western blot.7.NFE2L1-L gene affects SNAIL1 expression:The RNA and protein of BEAS-2B cells?NFE2L1-L-KD/Scramble?and 293T cells?NFE2L1-L-OE/Scramble?were collected.NFE2L1-L and SNAIL1 mRNA levels were detected by RT-qPCR.E-cadherin expression were detected at the protein level using western blot.8.Statistical analysis:All data in this study were analyzed using GraphPad Prism5software.Results1.Arsenic induced EMT and malignant transformation of human bronchial epithelial cells?BEAS-2B?:After the exposure to 0.1?mol/L,0.2?mol/L,0.5?mol/L NaAsO₂ for36 weeks,the level of E-cadherin mRNA in arsenic-treated cells was significantly lower than that of the vehicle control group?Veh?;the levels of SNAIL and TWIST2 mRNA were significantly higher than those of the Veh group,and the difference was statistically significant?P<0.05?.Protein level results showed that E-cadherin expression was significantly decreased in the 0.1?mol/L and 0.2?mol/L treatment groups,and Vimentin was significantly increased in the 0.2?mol/L and 0.5?mol/L treatment groups.The BEAS-2B cells treated with 0.1?mol/L NaAsO₂ for 36 weeks showed a significant effect on scratch healing after 48 hours of scratching compared with the control group.The number of meaningful clones in the groups exposed to chronic arsenic increased significantly compared with the control group,and the difference was statistically significant?P<0.05?.2.Inorganic arsenic exposure induced NFE2L1 expression in BEAS-2B cells:The expression of NFE2L1 was significantly increased by acute inorganic arsenic exposure,and the difference was statistically significant?P<0.05?.3.NFE2L1-L gene silencing:The mRNA level of NFE2L1-L in NFE2L1-L-KD group was significantly lower than that in scramble group,and the difference was statistically significant?P<0.05?.The NFE2L1 protein level in the NFE2L1-L-KD group was significantly lower than that in the SCR group regardless of As treatment group.4.NFE2L1-L silencing inhibited EMT and malignant transformation of BEAS-2B cells induced by arsenic:NFE2L1-L-KD cells were found to have significant difference in EMT index,scratch healing ability and/or clone formation ability compared with SCR cells at specific time points.After exposure to arsenic for 12 weeks,the mRNA and protein levels of E-cadherin in NFE2L1-L-KD cells were higher than those in SCR cells,and the SNAIL1 mRNA levels were decreased,compared with SCR cells?P<0.05?.Microscopically,NFE2L1-L gene-silenced cells were slightly stretched compared to SCR cells,similar to fusiform changes.Chronic arsenic exposure for 24 weeks resulted in significantly higher mRNA levels of SNAIL1 and TWIST2 in SCR cells than in the Veh group.The mRNA levels of SNAIL1 and TWIST2 in NFE2L1-L-KD cells were lower than those in the same treatment group?P<0.05?.The expression of E-cadherin in SCR cells in 0.1?mol/L treated group was significantly decreased.The level of Vimentin protein showed a positive correlation growth trend with arsenic dose.The expression of E-cadherin in NFE2L1-L-KD cells was higher than that in the same group of SCR cells.The SCR cells in the 0.1?mol/L arsenic treatment group were the first to have enhanced scratch healing ability.SNAIL1 and TWIST2 mRNA levels and E-cadherin protein expression were unchanged after 30 weeks of arsenic exposure compared with 24 weeks,while NFE2L1-L-KD cells showed an increase in SNAIL1 and a decrease in E-cadherin in the 0.5?mol/L treatment group?P<0.05?.There were more meaningful clones in SCR cells treated with 0.1?mol/L arsenic than that in Veh group?P<0.05?.The results of the scratch healing experiment showed that the scratch healing ability of the SCR cells in the 0.1?mol/L arsenic treatment group was still higher than that in the Veh group.36-week exposure to arsenic due to an increase of SNAIL1 and TWSIT2 mRNA in SCR cells.The same time,E-cadherin expression was decreased.NFE2L1-L-KD cells still had elevated SNAIL1 and decreased E-cadherin in the 0.5?mol/L treated group?P<0.05?.The SCR cells in the 0.1?mol/L arsenic treatment group were basically lost in the shape of paving stones,the cell boundaries were rough,and the intercellular connections were poor.The number of meaningful clones was significantly higher than that in the Veh group?P<0.05?.The scratch healing ability of SCR cells in the 0.1?mol/L arsenic treatment group was increased,compared with Veh group.5.NFE2L1-L gene regulates SNAIL1 expresssion:The mRNA level of SNAIL1 in NFE2L1-L-KD cells was decreased?P<0.05?.After the NFE2L1-L gene was silenced,the expression level of E-cadherin protein was significantly increased.293T cells stably transfected with NFE2L1-L overexpressing virus?NFE2L1-L-OE?have significantly higher mRNA levels of NFE2L1-L than SCR cell?P<0.05?.After treatment with acute EPO?20 nmol/L,6 h?,NFE2L1-L-OE cells had significantly higher levels of NFE2L1protein than SCR cells.With the overexpression of NFE2L1-L gene,SNAIL1 was increased in mRNA levels?P<0.05?.E-cadherin protein level in NFE2L1-L overexpressed cells was significantly lower than that in SCR group after exposure to EPO at 20 nmol/L for 6 h.Conclusion:1.Environmenrelevant doses of inorganic arsenic exposure induce malignant transformation and NFE2L1 expression in human bronchial epithelial cells.2.NFE2L1 silencing inhibits the malignant transformation of human bronchial epithelial cells induced by inorganic arsenic.3.NFE2L1 may participate in inorganic arsenic-induced cellular EMT by positively regulating SNAIL1 expression.