Effect Of Arsenic On The Expression Of Oncogene HER2 In Normal Bladder Epithelial Cells And Its Mechanism

Objective:Arsenic is a widely existing metal-like element in nature,which can enter the body through respiratory tract,digestive tract and skin,resulting in multiple organ and systematic damage.The arsenic pollution risk Assessment Model,developed by our college in cooperation with the Swiss Federal Water Research Institute,forecasts that the population exposed to high spirits in China may be as high as 20 million.Arsenic is a human carcinogen identified by the International Journal of Cancer Research,which can cause skin cancer,lung cancer and bladder cancer.Studies have shown that not only persistent arsenic exposure can cause tumors,but also the risk of bladder and lung cancer remains high decades after arsenic exposure has stopped.The proliferation rate of human normal bladder epithelial cells treated with low concentration sodium arsenite for a long time was significantly accelerated and malignant transformation was observed in our study group in vitro.Therefore,it is very important to explore the mechanism of arsenic-induced bladder cancer and to find effective prevention and control measures and treatment.Because arsenic itself does not cause point mutation,the study of its carcinogenic mechanism tends to be more likely to post-biochemical mechanism,that is,by disturbing the external genetic control of special sites,resulting in abnormal gene expression and carcinogenesis.Our previous study found that low-concentration arsenic can induce an increase in the expression of proto-oncogene epidermal growth factor receptor 2?HER2?in human normal bladder epithelial cells.HER2 is a member of the epidermal growth factor receptor?EGFR?family,and after activation,a plurality of signal paths related to cell proliferation,survival,invasion and angiogenesis can be activated,and are highly expressed in various tumors such as breast cancer,bladder cancer,gastric cancer,lung cancer and the like.HER2 plays an important role in the occurrence,development,invasion and metastasis of epithelial cancer.Therefore,if we can explore the regulation of arsenic-induced increase of HER2in bladder epithelial cells in its downstream tumorigenesis-related signaling pathway,it will help us to reveal the mechanism of arsenic-induced bladder cancer and provide scientific data for the prevention and treatment of bladder cancer in people exposed to high arsenic.Methods:In vivo experiment:sixty healthy F344 neonatal female rats?70-80g?were randomly divided into three groups with 20 rats in each group after one week of adaptation to the environment.The three groups were control group:drinking distilled water,50mg/L arsenite i As^IIIII exposure group and 200mg/L DMA^V exposure group.The rats in each group were killed after 12 weeks of free drinking water.The contents of arsenic in three treatment groups were determined by atomic absorption spectrophotometer.The proliferation of rat bladder epithelial cells was detected by HE staining,the mRNA expression of proliferation factor in rat bladder epithelial cells was detected by RT-PCR,and the protein expression of HER2activated by HER2 in rat bladder epithelial cells was detected by immunofluorescence and immunohistochemistry.The expression of EGF,TGF?,s E-cad in urine of rats was detected by ELISA.In vitro experiment:human normal bladder epithelial cell line?SV-HUC-1?was selected and purchased from the preservation center of typical American strains?ATCC,number:CRL-9520?.The cells were cultured in F12K complete medium?10%fetal bovine serum,100 U/ml penicillin,100?g/ml streptomycin?.The liquid was changed every other day and digested with 0.25%trypsin after the cells grew to85?90%fusion.The cells were cultured in F12K complete culture medium?10%fetal bovine serum,100 U/ml penicillin,100?g/ml streptomycin?.After the cells grew to 85?90%fusion,the cells were digested and passed down with 0.25%trypsin.Short-term arsenic-treated cell culture:After cell digestion and passage,inoculate the cells into a 60mm culture dish.When the cells enter the logarithmic growth stage,the concentration of NaAsO₂?0,1,2,4,8,10?M?.NaAsO₂ solution were prepared in F12k complete medium for 24h.The cells were treated with 10?g/ml EGF neutralizing antibody,5?g/ml TGF?neutralizing antibody,10?M GA?HSP90 inhibitor?,50 ng/ml EGF recombinant protein,5 ng/ml TGF?recombinant protein,50 ng/ml E-cad recombinant protein,10 ng/ml HSP90 recombinant protein,100 ng/ml IL-6 recombinant egg after pretreatment with E-cad or EGFR si RNA,for 4h.White and 10 ng/ml NDRG1 recombinant protein were pretreated for 30 min,and the cells were treated with short term arsenic.The mRNA expression level of HER2was detected by RT-PCR,and the protein expression level of HER2 activation was detected by immunofluorescence and Western Blot.The co-expression of HER2activation and companion protein HSP90 was detected by immunocoprecipitation.Long-term arsenic treatment cell culture:after digestion and passage,the cells were cultured in the complete culture medium without NaAsO2 for 24 h.After the cells were completely adherent to the wall and returned to the growth state,the culture medium was replaced with the complete culture medium containing 0.5?M NaAsO₂and continued to be cultured for 24 h.In this way,the malignant transformation model of bladder epithelial cells induced by arsenic was established by long-term culture for40 weeks,and the long-term culture control group was set up at the same time.The cells were treated with 30 nM NC negative control,EGFR or HER2 siRNA,were added with 10?g/ml EGF neutralizing antibody and 5?g/ml respectively.L TGF?neutralizing antibody,10?M GA?HSP90 inhibitor?,50 ng/ml E-cad recombinant protein,100 ng/ml IL-6 recombinant protein and 10 ng/ml NDRG1 recombinant protein were pretreated for 24 h.MTS assay was used to detect cell vitality,Transwell migration test and cell scratch test were used to detect cell migration ability.Western Blot technique was used to detect the expression of the above related factors activated by HER2.Detection of cell growth in cell culture medium by ELISA The expression level of EGF,TGF?,sE-cad.The data were analyzed by SPSS19.0 statistical software,and the experimental results were expressed by mean±standard deviation.Single factor variance analysis?ANOVA?was used to compare the statistical differences between the experimental group and the control group,and Dunnett's T3 test was used for the comparison between the two groups.T test was used to compare the two independent samples.There was significant difference at p<0.05.Result:1.Content of arsenic in urine of F344 ratsThe concentration and content of arsenic in 24 h urine of rats treated with arsenite and DMA^V were significantly higher than those of the control group.2.Long-term arsenite treatment leads to bladder epithelial hyperplasia in F344 rats.In the rats treated with DMA^V,the degree of hyperplasia of the bladder epithelial cells was significantly higher than that of the arsenate treatment group.The severity of cell proliferation in two groups was significantly higher than that in the control group.3.The effect of long-term arsenic treatment on the expression of EGFR and HER2 protein and phosphorylation of bladder epithelial cells in ratsCompared with the control group,the expression of EGFR and HER2 protein and phosphorylation in bladder cells of rats treated with arsenite and DMA^V were significantly increased,but there was no significant difference between the two groups.4.Effects of long-term arsenic treatment on the expression of EGF,TGF?,E-cad and sE-cad in rat bladder epithelial cellsCompared with the control group,the expression of EGF,TGF?protein and E-cad protein in bladder epithelial cells of the two arsenic treatment groups were significantly higher than those of the control group.The expression of EGF and E-cad in DMA^V treatment group was significantly different from that in arsenite group.5.Effect of long-term arsenic treatment on the expression of HSP90,IL-6,NDRG1 protein in rat bladder epithelial cellsCompared with the control group,the expression of HSP90 protein in bladder epithelial cells of rats treated with arsenic was significantly increased,the expression of NDRG1 protein was significantly decreased,and the expression level of IL-6protein remained unchanged.Compared with arsenite group,the expression of HSP90protein in DMA^V treatment group was significantly different from that in arsenite group.6.Effect of long-term arsenic treatment on the expression of proliferating factor protein in rat bladder epithelial cellsCompared with the control group,the mRNA expression of CCND,COX-2 and protein expression of CCND,COX-2,PCNA,KI67 in bladder epithelial cells of the two arsenic treatment groups were significantly higher than those of the control group,while the expression of CCND,COX-2,PCNA,KI67 protein was significantly higher than that of the control group.7.Effect of short-term sodium arsenite exposure on HER2 expression and phosphorylation in SV-HUC-1 cellsShort-term arsenite could stimulate HER2 phosphorylation in SV-HUC-1 cells,and the phosphorylation level of HER2 reached the highest level when treated with 2?M arsenite for 24 h.8.Effects of short-term sodium arsenite exposure on EGFR expression and phosphorylation in SV-HUC-1 cellsShort-term arsenite could stimulate EGFR phosphorylation in SV-HUC-1 cells,and the phosphorylation level of EGFR reached the highest level when treated with 2?M arsenite for 24 h.It is completely consistent with the phosphorylation trend of HER2.9.In SV-HUC-1 cells treated with sodium arsenite,EGFR is involved in the activation of HER2 phosphorylationSodium arsenite activated HER2 depends on the activation of EGFR,and EGFR is involved in the activation of HER2.10.Effect of short-term sodium arsenite on the expression of extracellular growth factor in SV-HUC-1 cellsThe levels of EGF,TGF?and sE-cad in the arsenic-treated bladder epithelial cells were significantly higher than those in the control group,and the protein level of E-cad was significantly lower than that of the control group.11.EGF is involved in the activation of HER2 phosphorylation in SV-HUC-1cells treated with sodium arsenite for a short period of time.EGF is able to stimulate the phosphorylation of HER2 in the bladder epithelial cells treated with arsenite.12.In SV-HUC-1 cells treated with sodium arsenite in the short term,TGF?is involved in the activation of HER2 phosphorylationIn the bladder epithelial cells treated with arsenite,the TGF?is capable of stimulating the phosphorylation of HER2.13.In SV-HUC-1 cells treated with sodium arsenite,E-cad was not involved in the activation of HER2 phosphorylationIn arsenite-treated bladder epithelial cells,E-cad and HER2 phosphorylation are inhibited from each other,and the decrease of E-cad is not the cause of HER2phosphorylation,but HER2 phosphorylation can inhibit E-cad.14.Effect of short-term sodium arsenite exposure on HSP90,NDRG1,IL-6expression in SV-HUC-1 cellsIn arsenic treated bladder epithelial cells,the level of HSP90 protein was significantly higher than that of the control group,and the protein level of NDRG1,IL-6 was significantly lower than that of the control group.15.Effect of HSP90 on phosphorylation of HER2 in SV-HUC-1 cells treated with sodium arsenite for a short period of timeIn arsenite treated bladder epithelial cells,HSP90 interacts with phosphorylated HER2 as a molecular chaperone protein.16.Effect of IL-6 and NDRG1 on the phosphorylation of HER2 in SV-HUC-1 cells treated with sodium arseniteIn arsenite-treated bladder epithelial cells,NDRG1,IL-6 were negatively correlated with p-HER2 levels.In addition,increased p-HER2 levels in arsenic exposed SV-HUC-1 cells may be due to the downregulation of NDRG1 protein levels after arsenite inhibits IL6 expression.17.Effects of long-term arsenic treatment on the activity and proliferation migration of SV-HUC-1 cells.The cell viability and proliferation of SV-HUC-1 cells were significantly higher than that in the control group after 40 weeks of treatment with 0.5?mol/L NaAsO₂.18.Effects of long-term arsenic treatment on expression of SV-HUC-1cell-related cytokine protein.After treated with 0.5?mol/L NaAsO2 for 40 weeks,the phosphorylation of EGFR and HER2,the protein expression of TGF?,HSP90,proliferating factor COX2,PCNA and CCND increased significantly,the expression of IL-6 and NDRG1 protein decreased significantly,and the expression of TGF?,EGF and sE-cad protein also increased significantly in SV-HUC-1 cells treated with 0.5?mol/L NaAsO₂ for 40weeks.However,the total protein expression of EGFR and HER2 remained unchanged.19.Effects of EGFR and HER2 on proliferation and migration of SV-HUC-1cells treated with arsenite for a long timeThe knockdown of EGFR and HER2 can significantly inhibit the proliferation and migration of long-term arsenite-treated cells.20.Effects of related factors involved in EGFR and HER2 activation in SV-HUC-1 cells treated with arsenite for a long timeIn SV-HUC-1 cells treated with arsenite,HER2 may be activated by a heterodimerization with EGFR.Due to the activation of EGFR and HER2,the expressions of E-cad,sE-cad,NDRG1,IL-6,COX2,CCND and PCNA were changed,while the up-regulated expressions of TGF?,EGF and HSP90 might not be mediated by the activation of EGFR and HER2.21.Activation of EGFR and HER2 phosphorylation by EGF,TGF?and HSP90 in SV-HUC-1 cells treated with arsenic for a long timeTGF?,EGF and HSP90 were involved in the phosphorylation of EGFR and HER2 in arsenite treated cells.22.The effects of E-cad,IL-6,NDRG1 on the phosphorylation of EGFR and HER2 in SV-HUC-1 cells were inhibited by long-term arsenic treatment.Both IL-6 and NDRG1 can regulate the phosphorylation of EGFR and HER2 in arsenite treated cells.And the process is reversible.Conclusion:1.Both in vitro and in vivo experiments showed that low dose arsenic treatment could induce the proliferation of normal bladder epithelial cells for a long time.2.Animal experiment:the expression of EGF,TGF?,sE-cad,HSP90,EGFR,HER2,p-EGFR,p-HER2,proliferation factor PCNA,CCND and COX2 increased,IL-6 remained unchanged and NDRG1,E-cad expression decreased in arsenite exposed group for 12 weeks.3.Cell experiment:The expression of EGF,TGF?,sE-cad,HSP90,p-EGFR,p-HER2 and proliferation factor PCNA,CCND and COX2 increased in the short and long-term arsenic-containing bladder epithelial cells,and the expression of HE1 and HER2 was unchanged;and the expression of IL-6,NDRG1 and E-cad decreased.4.TGF?,EGF and HSP90 are involved in the regulation of phosphorylation of EGFR and HER2 in arsenite treated cells,which is an irreversible regulatory effect.5.IL-6,NDRG1 can inhibit the phosphorylation of EGFR and HER2 in arsenite treated cells,which is also a reversible mutual regulation.