NF-kB-induced EMT Via IL-6 Is Involved In Cigarette Smoke Extract-induced Cell Malignant Transformation

Cigarette smoking constitute a major health hazard within the scope of the world,epidemiological evidences suggest that there is correlation between exposure to cigarette smoke and the crowd incidence of lung cancer.The causal relationship between smoking and the pathological changes of lung or other organs has been confirmed by a number of studies.Lung cancer is one of the most common malignant tumor in the world which has great threat to human health.Although researches about the relationship between cigarette smoking and lung cancer has become more widely,most of them are studies about epidemiology instead of molecular mechanisms.Altough there are sudies confirmed that smoking is the primary risk factor of lung cancer,the molecular mechanisms of cigarette smoking-induced lung cancer remains to be further study.Long-term exposure to cigarette smoke may cause lung damage and inflammation reaction,which promote the development of lung cancer.Signaling pathways mediated by inflammatory cytokines are involved in the evolution of malignant tumor cells.Epithelial-mesenchymal transition(EMT)is a key mechanism of the evolution process of malignant tumor,so the EMT is considered to be a bridge between inflaunation and cancer.In the process of EMT and cancer metastasis,many miRNAs related to inflammation become the link between EMT and inflammation,miR-200 family can maintain the epithelial cell phenotype and regulate the EMT proeess through targeted regulation of ZEB1 和 ZEB2 expressions,which are transcription repression factors of E-cadherin.The important roles of cigarette smoke-induced NF-κB activation in the process of EMT,malignant transformation and tumorigenicity through regulating inflammation reactions and miR-200c expressions have not been reported before.Therefore,this study constructed the malignant transformation model of human bronchial epithelial cells which was induced by CSE chronic treatment.Based on this model,a variety of molecular biology methods is applied to evaluate the effects of NF-κB activation on IL-6 and miR-200c levels.Our study try to reveal the important role of NF-κB in inflammation and EMT process during CSE-induced malignant transformation and tumorigenicity in HBE cells.This study provides clues for the molecular mechanisms of cigarette smoking-induced lung cancer,which will provide some new clues to find biomarkers and prevention measures of CSE-induced lung cancer.Methods1.Effects of cigarette smoke extract treatment on the cell toxicity,malignant transformation and tumorigenicity of HBE cellsWe detected the cell toxicity with CCK8 experiment.HBE cells were exposed to CSE(0,5,10,20,50,or 100 μg/mL)for 24 or 48 h.And the concentrations of 0 or 20 μg/mL CSE were chosen to treat HBE cells for about 40 passages(20 weeks),two days to extend a passage.We observed the degree of cell malignant with soft agar colony experiment,and tested cell tumorigenicity with nude mice subcutaneously into tumor experiment.We are going to observe the roles of CSE on the cell toxicity,malignant transformation and tumorigenicity of HBE cells.2.Effects of cigarette smoke extract treatment on the epithelial-mesenchymal transition of HBE cellsWhen HBE cells exposed to 0 or 20μg/mL CSE for 0、20、30 and 40 passages,we observed cell morphology under inverted microscope and detected the levels of EMT marker proteins with Western blot and immunofluorescence test.0 or 20μg/mL CSE were used to treat HBE cells for about 40 passages(20 weeks);HBE cells also exposed to CSE over periods ranging from 0 to 24 h,and miR-200c levels were determined in HBE cells with qRT-PCR.We are going to observe the effects of CSE treatment on the EMT of HBE cells.3.Effects of cigarette smoke extract on IL-6 levels of HBE cellsHBE cells were exposed to CSE(0 or 20μg/mL)for 40 passages(20 weeks),or exposed to 0 or 20 μg/mL CSE for 0,6,12,or 24 h.RT-PCR and ELISA were used to detect the IL-6 expressions and secretions respectively.We are going to observe the effects of CSE on IL-6 levels of HBE cells.4.The roles of IL-6 involved in cigarette smoke extract-induced downregulation of miR-200c levels and cell malignant transformation in HBE cellsHBE cells were treated by 0.5μg/mL anti-IL-6 neutralizing antibody or anti-IgG antibody combined with 0 or 20μg/mL CSE or 24 h.ELISA were used to detect the IL-6 secretions and miR-200c levels were determined with qRT-PCR in HBE cells.We also detected the capacity of colony formation by HBE cells which were exposed to 0 or 20μg/mL CSE for 40 passages with or without IL-6 by use of anti-IL-6 neutralizing antibody.We are going to observe the roles of IL-6 involved in CSE-induced downregulation of miR-200c levels and cell malignant transformation in HBE cells.5.Effects of cigarette smoke extract on NF-κB activationHBE cells were exposed to CSE(0 or 20μg/mL)for 40 passages(20 weeks),or exposed to 20 μg/mL CSE for 0,6,12 or 24 h.Western blot were used to detect the NF-κB p65 levels and its phosphorylation levels.We are going to observe the effects of CSE on NF-κB activation.6.The roles of NF-κB in cigarette smoke extract induced upregulation of IL-6 levels and downregulation of miR-200c levels in HBE cellsHBE cells were pretreated with 10μM NF-λB inhibitors Bay11-7082,then added 0 or 20 μg/mL CSE processing 24 h.Westerm blot were used to detect the the NF-κB p65 levels and its phosphorylation levels,the distribution of NF-κB p65 in the cells was tested by immunofluorescence test.RT-PCR and ELISA were used to detect the IL-6 expressions and secretions respectively,miR-200c levels were determined with qRT-PCR;Or pretreated HBE cells with 10 NF-κB inhibitors Bay11-7082 for 3 h,then added 10 ng/mL human recombinant cytokines IL-6 combined with 0 or 20μg/mL CSE processing 24 h,we determined miR-200c levels with qRT-PCR.We are going to observe the roles of NF-κB in CSE induced upregulation of IL-6 levels and downregulation of miR-200c levels in HBE cells.7.The roles of NF-κB in the upregulation of IL-6 induced by cigarette smoke extract chronic treatmentWith 10 μM NF-κB inhibitors Bayll-7082 pretreatment for 3 h,then added 0 or 20 μg/mL CSE processing 24 h.Went down to the future generation every 2 days,continue to treat with CSE after Bayll-7082 pretreatment.HBE cells were treated according to this way until 40 passages(20 weeks).Western blot were used to detect the the NF-κB p65 levels and its phosphorylation levels,RT-PCR and ELISA were used to detect the IL-6 expressions and secretions respectively.We are going to-observe the roles of NF-κB in the upregulation of IL-6 induced by cigarette smoke extract chronic treatment.8.The roles of NF-κB involved in the changes of EMT marker proteins and the malignant transformation induced by cigarette smoke extract chronic treatment in HBE cellsPretreated with 10 μM NF-κB inhibitors Bay 11-7082 for 3 h,then added 0 or 20 μg/mL CSE processing 24 h,went down to the future generation every 2 days,continue to treat with CSE after Bay11-7082 pretreatment.HBE cells were treated according to this way until 40 passages(20 weeks).We detected the levels of EMT marker proteins with western blot,determined the miR-200c levels with qRT-PCR.And the malignant degrees of HBE cells were tested with soft agar colony experiment.We are going to observe the roles of NF-κB involved in the changes of EMT markerproteins and the malignant transformation induced by CSE chronic treatment in HBE cells.Results1.Effects of cigarette smoke extract treatment on the cell toxicity,malignant transformation and tumorigenicity of HBE cellsThe results showed that 50 and 100 μg/mL CSE has obvious toxic effects on the HBE cells,concentration of 20 μg/mL and less than 20 μg/mL CSE has no obvious toxic effect on the HBE cells.HBE cells exposed to 20 μg/mL CSE for about 20 weeks formed more colonies than passage control cells in soft agar.The mice injected with CSE-transformed HBE cells formed tumors.And pathology biopsy showed low differentiation epithelioid-liked cancer cells.These results suggest that CSE induce the malignant transformation and tumorigenicity of HBE cells.2.Effects of cigarette smoke extract treatment on the epithelial-mesenchymal transition of HBE cellsOur data showed that in cells chronically exposed to 20 μg/mL CSE,with the increased malignant degrees,the cells gradually acquired a fibroblast-like,mesenchymal appearance consistent with EMT.The levels of Epithelial marker E-cadherin levels and its fluorescence intensity were decreased gradually;in contrast,N-cadherin,vimentin levels and the fluorescence intensity were increased.Furthermore,both acute and chronic treatment with 20μg/mL CSE downregulated miR-200c levels.These results suggest that chronic exposure of HBE cells to CSE undergo an EMT.3.Effects of cigarette smoke extract on IL-6 levels of HBE cellsOur results found that in the malignant transformation HBE cells induced by 20μg/mL CSE chronic treatment until 40 generations,both mRNA levels and secretion levels of IL-6 were siguificantly elevated;and HBE cells with acute treatment of 20μg/mL CSE,IL-6 mRNA levels and secretion levels were significantly increased in 12 h and 24 h,with a certain amount of time effect relation.These resultes suggest that CSE induce inflammation response through upregulating IL-6 levels in HBE cells.4.The roles of IL-6 involved in cigarette smoke extract-induced downregulation of miR-200c levels and cell malignant transformation in HBE cellsThe results showed that anti-IL-6 neutralization led to restored expressions of CSE-upregulated IL-6 secretion levels,blocked expressions of CSE-downregulated miR-200c levels.In CSE-transformed HBE cells,depleted of IL-6 by the antibody displayed fewer colonies compared with the untreated group.These data show that IL-6 was involved in CSE-induced downregulation of miR-200c levels and cell malignant transformation.5.Effects of cigarette smoke extract on NF-κB activationOur data showed that in the malignant transformation HBE cells induced by 20μg/mL CSE chronic treatment until 40 generations,p-NF-κB p65 levels was significantly elevated;and HBE cells with acute treatment of 20 μg/mL CSE,P-NF-μB p65 levels was significantly increased in 12 h and 24 h,with a certain amount of time effect relation.These data indicate that CSE treatment activate NF-κB in HBE cells,which prompt that NF-κB may play an impartant role in CSE-induced cell transformation.6.The roles of NF-κB in cigarette smoke extract induced upregulation of IL-6 levels and downregulation of miR-200c levels in HBE cellsResults show that block the activation of NF-κB with Bay11-7082 was able to block CSE-induced upregulation of protein levels and the nuclear transfer of p-NF-κB p65 proteins,the IL-6 levels and the downregulation of miR-200c levels.Raise IL-6 levels after inhibiting the activation of NF-κB can reverse the rise of miR-200c levels.Our results prompt that the activation of NF-κB may play an important role in CSE-induced upregulation of IL-6 levels and downregulation of miR-200c levels in HBE cells.7.The roles of NF-κB in the upregulation of IL-6 induced by cigarette smoke extract chronic treatmentThe results showed in the malignant transformation HBE cells induced by 20μg/mL CSE chronic treatment until 40 generations,p-NF-κB p65 levels,IL-6 mRNA levels and secretion levels were significantly elevated;however,long-term treatment with the NF-κB inhibitors Bay11-7082 can block CSE chronic treatment induced NF-κB activation and IL-6 levels.These data indicate that NF-κB activation play an important role in the upregulation of IL-6 induced by CSE.8.The roles of NF-κB involved in the changes of EMT marker proteins and the malignant transformation induced by cigarette smoke extract chronic treatment in HBE cellsThe results showed that NF-κB inhibitors long-term treatment inhibited the downregulation of E-cadherin and miR-200c levels and the upregulation of N-cadherin and viynentin levels induced by 20μg/mL CSE.Futher,NF-κB inhibitors inhibited the soft agar colony formation ability of CSE-transformed HBE cells.Our results indicate that the activation of NF-κB in HBE cells plays an important role in in the changes of EMT marker proteins and the malignant transformation induced by CSE.Conclusions1.CSE chronic treatment can induce EMT,the malignant transformation and tumorigenicity of HBE cells.2.CSE chronic treatment can induce inflammation in HBE cells,and inflammatory cytokines IL-6 play an important role in CSE-induced EMT and the malignant transformation.3.NF-kB-induced EMT via IL-6 plays an important role in cigarette smoke extract-induced cell malignant transformation.