The Study Of Ca²⁺/NFAT2 And NF-?B Signaling Pathway Promoting Proliferation And Inflammatory Factor Expression In Malignant Transformation Induced By Arsenic In Human Uroepithelial Cells

Objective:Arsenic is a potent human carcinogen whose primary target organs are the urinary bladder,lung and skin.Due to the great diversity of biological effects of arsenic,animal carcinogenesis models have not yet been directly replicated.The mechanism of carcinogenesis has been focused on arsenic-induced malignant transformed cells.More and more studies have shown that the inflammatory response caused by environmental chemicals plays an important role in the malignant transformation and carcinogenesis of cells.At present,the inflammation-cancer transformation mechanism has become a research hotspot.In the study of numerous effect mechanisms,it has been found that long-term chronic inflammation causes the defensive function of the body to collapse and can induce various types of tumors.Inflammation plays an important role in all stages of tumor development and there is a complex relationship between them.Arsenic can induce the production of inflammatory cytokines and induce epithelial mesenchymal transition?EMT?in the arsenic-induced malignant transformation of cells,eventually resulting in the malignant transformation of cells.Nuclear factor of activated T cells?NFAT?was originally found as a transcription factor that binds to the activated T cell IL-2 promoter.It has been found that members of the NFAT family continued to be activated and/or overexpressed in a variety of tumor cells.NFAT-regulated genes play an important role in cell cycle,cell development and differentiation,tumorigenesis,angiogenesis and apoptosis,suggesting that NFAT protein is potentially tumorigenic.NF-?B is an important nuclear transcription factor that regulates inflammation and tumorigenesis in the form of homodimers or heterodimers,of which p50/p65 is the most prevalent and most versatile NF-?B protein form.Almost all of the genes involved in inflammation are regulated by activated NF-?B.In a variety of tissue tumors,including bladder cancer,NF-?B activity was significantly increased in the tumor,which has played a key role.NF-?B signaling pathway affect the survival,proliferation,differentiation,death of cells and many other biological processes.Therefore,this study intends to establish arsenic induced malignant transformation of uroepthelial cells,investigate the expression of tumor-promoting factors and inflammatory factors during malignant transformation of cells,and the activation of NFAT and NF-?B signaling pathways,and their role in promoting the expression of tumor-associated factors and inflammatory factors.Methods:The SV40 immortalized human uroepithelial cell line?SV-HUC-1?was purchased from the American Type Culture Collection?ATCC,Wiltshire,USA?.Cells were cultured in F-12 medium supplemented with 10%FBS,100 U/ml penicillin and100?g/ml streptomycin.Cells were grown in T-75 culture flasks at 37°C in a humidified 5%CO₂ incubator.SV-HUC-1 cells were grown to 80%confluence for all treatments.Cells were either exposed to different concentrations of NaAsO2,or were pretreated with PDTC?50?mol/L?,CsA?10?mol/L?,U0126?10?mol/L?,SB203580?10?mol/L?,SP600125?10?mol/L?,LiCl?10 mM?before NaAsO₂exposure.All treatments were performed in triplicate as indicated.Long-term arsenic treatment of SV-HUC-1,the cells were digested and subcultured,and cultured in a complete medium without NaAsO₂ for 24 hours.After the cells were completely adherent,and the growth state was restored,the culture solution was changed to a complete culture solution containing 0.5?mol/L NaAsO₂.Continue to cultivate 24-48h.Such repeated,long-term cultivation,while the establishment of long-term culture of non-arsenic treatment group.Short-term arsenic treatment for SV-HUC-1:after cell digestion and passage,the cells were seeded into 60 mm culture dishes until the cells entered the logarithmic growth phase,and exposed to a solution of NaAsO₂?0,1,2,4,8,and 10?mol/L?in F12K complete medium for 24 h.Three or more parallel samples were set for each set of experiments.MTS method was used to detect cell viability,cell scratches and transwell migration assays were used to test the cell migration ability,and soft agar colony formation assay was used to detect the cell malignancy,plate clone formation assay was used to test cell proliferation.In our study,Fluo-3,AM dye and flow cytometry were used to detect intracellular Ca²⁺levels.RT-qPCR and Western Blot techniques were used to detect the mRNA and protein expression levels of the key factors of NFAT2 and NF-?B signaling pathway.RT-qPCR and Western Blot techniques were used to detect mRNA and protein expression of pro-angiogenic factors?Bcl2,CCND,PCNA,MMP1?and inflammatory factors?TNF?,TGF?,TGF?1,IL8,and IL1??,respectively.The data were analyzed using the SPSS 17.0 software.The Kolmogorov–Smirnov test was used to evaluate the normality of the data.Normally distributed and logarithmic transformation normalized distribution variables were analyzed with parametric tests.Differences between two groups were analyzed by Student's unpaired t test.One-way analysis of variance?ANOVA?followed by least significant difference?LSD?or Dunnett's T3 test was used to determine differences among groups of arsenic-treated SV-HUC-1 cells.Result:1 The effects of long-term arsenic treatment on cell viabilityWith the prolongation of arsenic treatment,cell viability increased significantly.The viability of cells treated with arsenic for 30 weeks and 40 weeks was 194%and210%of cells in the control group,respectively.2 Cell scratches,cell migrationAfter long-term arsenic-treated cells,the scratch healing ability is greatly enhanced,and with the extension of arsenic time,the scratch healing rate significantly accelerated.Transwell experimental results showd that long-term arsenic-treated cells migrate significantly enhanced.3 Soft agarAfter long-term arsenic-treated cells,colony formation on soft agar was observed,indicating that SV-HUC-1 cells undergo malignant transformation after being treated with 0.5?mol/L NaAsO₂ for 40 weeks.4 Effects of long-term arsenic treatment on mRNA and protein expression of tumor related factorsThe mRNA and protein expressions of Bcl2,CCND,PCNA,MMP1 in non-arsenic-treated cells did not change.With the NaAsO₂ treatment,the mRNA and protein expression of Bcl2,CCND,PCNA and MMP1 were increased,and their expression levels gradually increased with the prolonged exposure time.5 Effects of long-term arsenic treatment on mRNA and protein expression of inflammatory cytokinesThe mRNA and protein expressions of TNF?,TGF?1,IL8 and IL1?in non-arsenic treated cells did not change.With the NaAsO₂ treatment,the m RNA and protein expressions of TNF?,TGF?1,IL8 and IL1?increased,with the extension of time,the expression level gradually increased.6 Effect of sodium arsenite on the viability of SV-HUC-1 cellsThe cell viability of NaAsO2-treated group was decreased at all doses,and the higher the dose of NaAsO2,the lower the cell viability.When the concentration reached 4?mol/L,the cell viability had dropped to 80%.When the concentration reached 10?mol/L,the cell viability had dropped to 60%.7 The level of intracellular Ca²⁺levelsWhen the concentration reached 8 and 10?mol/L,the intracellular calcium level in SV-HUC-1 cells was increased.8 Effect of short-term arsenic treatment on the expression of NFAT2 mRNA and proteinThe mRNA and protein expression of NFAT2 did not change in each treatment group,p-NFAT2 expression decreased.The nuclear expression of NFAT2 increased under the treatment of 2-10?mol/L,and the expression in 4?mol/L treatment group was the highest.9 The expression and activation of NFAT2 in long-term arsenic treatment SV-HUC-1 CellsThe m RNA expression of NFAT2 was not changed in the non-arsenic treatment group,while the mRNA expression of NFAT2 was increased in the 40-week treatment with 0.5?mol/L NaAs O₂.Consistent with the mRNA expression,the protein expression of NFAT2 was not changed in the non-arsenic treatment group;after 40weeks of arsenic treatment,the NFAT2 protein expression was increased.After 20weeks of treatment with 0.5?mol/L NaAsO2,nuclear NFAT2 protein expression increased.10 ERK/MAPK is involved in NFAT2 activationERK inhibitor?U0126?significantly reduced the nuclear expression of NFAT2,while JNK and p38 inhibitors was not blocked NFAT2 nuclear translocation induced by arsenite.11 GSK3?is involved in NFAT2 activationThe expression of GSK3?in the nucleus did not change,while the expression of p-GSK3??Ser9?increased significantly.GSK3?inhibitor?Li Cl?promotes NFAT2dephosphorylation and activation into the nucleus.12 Effect of NFAT inhibitor?CsA?on the expression of tumor related proteinThe inhibition of activation of NFAT2 pathway significantly reduced the protein expression of Bcl2,CCND,and PCNA protein expression.13 Effect of NFAT inhibitor?CsA?on mRNA and protein expression of inflammatory cytokinesNFAT2 signal pathway inhibitor can significantly inhibit arsenic-induced expression of cytokines TNF?,TGF?,TGF?1,IL8 mRNA and protein levels.14 The cell proliferation in arsenic-induced malignant transformation cell depends on persistent activation of NFAT2Cells treated with arsenic for 40 weeks formed clones,whereas NFAT2 inhibitors significantly inhibited clonogenicity of transformed cells and inhibited the ability of transformed cells to proliferate in vitro.15 Effects of short arsenic treatment on IKK?/I?B?/NF?B signaling pathway in SV-HUC-1 cellsThe mRNA level of IKK?decreased in 8 and 10?mol/L treatment groups,while the mRNA expression of IKK?increased in 2?mol/L treatment group and decreased in 10?mol/L treatment group.The level of IKK?protein did not change below 4?mol/L NaAsO₂,and decreased when NaAsO₂ concentration was higher than8?mol/L.Under low dose of NaAsO₂ treatment?2 and 4?mol/L?,the expression level of IKK?protein increased and decreased at 10?mol/L,which was consistent with the result of mRNA.Compared with the control group,the expression of I?B?slightly decreased after treatment with 4?mol/L sodium arsenite.Under the condition of 2-10?mol/L,p65 was activated into the nucleus and its nuclear protein expression was significantly increased,and the highest expression level was found at 4?mol/L.Under the condition of 1-4?mol/L,p50 activated into the nucleus The nuclear protein expression level was significantly higher than that of the control group,and the expression level was highest in the 4?mol/L treatment group.16 Effects of long-term arsenic treatment on NF?B signaling pathway in SV-HUC-1 cellsThe mRNA and protein level of IKK?and IKK?in non-arsenic-treated cells did not change.However,the mRNA and protein expression of IKK?and IKK?increased after prolonged treatment with 0.5?mol/L NaAsO₂ for 30 weeks,with the extension of exposure time,the expression level gradually increased.Compared with the control group,the IKK?and IKK?protein expression levels in the arsenic-treated 30 and 40weeks increased by 156%and 169%,respectively,182%and 224%.The protein expression of NF-?B p65,p50 in nuclei were significantly increased under long-term treatment with sodium arsenite.The expression level of arsenic-treated nuclei at 30and 40 weeks was significantly higher than that of the control group.17 MAPK participates in the activation of NF-?B signaling pathwayERK and JNK inhibitors blocked p65 and p50 phosphorylation and reduced the expression of p50 and p65 nuclear proteins.18 Effect of NF?B signaling pathway on the expression of tumor related proteinThe inhibition of activation of NF kappa B pathway can significantly reduce the expression level of Bcl2,Cycllin D1,PCNA.19 Effect of NF-?B signaling pathway on the expression level of inflammatory factors mRNA and proteinAfter the activation of NF?B pathway was blocked,the mRNA and protein expressions level of TNF?,TGF?,TGF?1 and IL8 were significantly decreased.20 Cell Proliferation in arsenic-induced malignant transformed cells depends on the sustained activation of NF-?BInhibitors of NF-?B can significantly inhibit colony formation of transformed cells and inhibit the in vitro proliferation of transformed cells.21 The interact effect between NF-?B and NFAT2The NF-?B signaling pathway is partially regulated by CaN and inhibits activation of the NFAT2 signaling pathway.Conclusion:1 Long-term and low-dose arsenic treatment induced malignant transformation of human normal bladder epithelial cells.2 In arsenic-induced malignant transformation cell,the mRNA and protein expression of Bcl2,Cyclin D1,MMP1 and PCNA were increased,and the m RNA and protein expression of TNF?,TGF?,TGF?1,IL8,IL1?mRNA and protein were increased.3 Arsenic induced NFAT2 activation into the nucleus,activated the Ca²⁺/NFAT2signaling pathway.ERK is involved in NFAT2 activation;GSK3?negatively regulates NFAT2 signaling pathway.4 Arsenic activated IKK?/I?B?/NF-?B signaling pathway,p65 and p50translocated into the nucleus;ERK and JNK participate in the activation of NF-?B signaling pathway.5 Interaction between NFAT2 and NF?B signaling pathway promotes cell proliferation;and induced the inflammatory factors?TNF?,TGF?,TGF?1,IL8?expression.