Study On The Role Of Wnt/?-catenin Signaling Pathway In Atmospheric PM_2.5 Induced P16 Gene Methylation And Cellular Biological Behavior Changes

ObjectiveAs one of the air pollutants,atmospheric PM_2.5.5 is obviously associated with the occurrence and development of lung cancer.To observe the role of Wnt/?-catenin signaling pathway in the methylation alteration of p16 tumor suppressor gene promoter in bronchial epithelial cells?Beas-2B?and non-small cell lung cancer cells?A549?caused by atmospheric PM_2.5.To explore the role of Wnt/?-catenin signaling pathway in the changes of biological behavior of Beas-2B and A549 cells induced by atmospheric PM_2.5.The aim of this study was to investigate the molecular mechanism of PM_2.5.5 on lung cancer from the perspective of carcinogenesis and cancer promotion by in vitro culture of normal bronchial epithelial cells and lung cancer cells.MethodsSamples were collected according to the procedures described in our group's previous studies.After drying and weighing,the extract was dissolved in DMSO to a concentration of 100 mg/mL for use.Beas-2B and A549 cells were incubated with different does of atmospheric PM_2.5.5 for 12 h.MTT,cell scratch test and the transwell invasion assay were used to confirm the concentration of the drug.MTT assay was used to determine the effect of atmospheric PM_2.5.5 on cell viability,and to confirm the optimal concentration of inhibitors.The levels of proteins involved in Wnt/?-catenin signaling pathway,DNMT1 and EMT related protein were measured by western blot;The levels of mRNA and DNA involved in Wnt/?-catenin signaling pathway and the mRNA level of DNMTS were measured by RT-qPCR;The methylation specific PCR?MSP?assay was used to assess the methylation level of p16 tumor suppressor gene promoter;Cell scratch test to detect the change of migration level of A549 cells after co-incubation with inhibitor DKK-1 and PM_2.5;After cells were exposed to different does of atmospheric PM_2.5,as well as DKK-1+PM_2.5,the transwell invasion assay was used to assess the invasiveness of A549 cells.ResultsThe role of Wnt/?-catenin pathway in atmospheric PM_2.5.5 induced p16 methylation and cellular biological behavior changes in A549 cells1.MTT assay was used to determine the dose of PM_2.5.5 and DKK-1.At the dose of 100?g/mL for 24 h after treatment,the cell survival rate was close to 81%,which was statistically significant compared with the control group?P<0.01?.Therefore,the dose selected in this study included 0,25,50,100?g/mL,and the exposure time was set at24 h.With the increase of DKK-1 concentration,the cell survival rate showed a declining trend.When the dose of DKK-1 was 50 ng/mL,the cell survival rate was 94%,showing no statistical difference compared with the control group.1.PM_2.5.5 causes the increase of cell methylation level.Compared with the control group,DNMT1 protein expression level and p16 gene methylation level increased as the dose increased,and the effect was strongest at the concentration of 100?g/mL?P<0.01?.3.PM_2.5.5 causes changes in cellular biological behavior.Compared with the control group,cell migration was significantly increased when the concentration was 25,50?g/mL?P<0.01?,and no significant change was observed when the concentration continued to rise to 100?g/mL.Compared with the control group,the invasiveness of cells was enhanced in the 25?g/mL group,but significantly decreased as PM_2.5concentration continued to rise to 50,100?g/mL?P<0.01?.Therefore,the cells were divided into four groups?0,6.25,12.5,25?g/mL?to detect the EMT protein level.Compared with the control group,Vimentin protein expression level was up-regulated and E-cadherin protein expression level was decreased with the increase of exposure dose?P<0.01?.4.PM_2.5.5 activates Wnt/?-catenin signal pathway.With the increase of the dose,the expression levels of p-GSK-3?/GSK-3?,?-catenin and p-GSK-3?protein were up-regulated,while the expression levels of APC protein were decreased.5.DKK-1 inhibits the Wnt/?-catenin signal pathway activated by PM_2.5.Compared with the single exposure group,the p-GSK-3?/GSK-3?ratio and the expression levels of?-catenin and p-GSK-3?protein in the co-incubation group of PM_2.5.5 and DKK-1decreased,while the expression levels of APC protein were up-regulated,which were statistically significant?P<0.01?.6.DKK-1 inhibited p16 methylation and cellular biological behavior changes after PM_2.5.5 activation of Wnt/?-catenin signaling pathway.Compared with the poisoned group alone,the DNMT1 protein expression level and p16 methylation level were decreased in the PM_2.5.5 and DKK-1 co-incubation group,as well as the cell migration and inventiveness,the Vimentin expression level was decreased,and the E-cadherin protein expression was up-regulated?P<0.01?.Wnt/?-catenin pathway plays a role in causing changes in p16 methylation and EMT protein expression levels of Beas-2B cells induced by atmospheric PM_2.51.MTT assay was used to determine the dose of PM_2.5.5 and DKK-1.After 24 h of treatment with 100?g/mL,the cell survival rate was close to 75%,which was statistically significant compared with the control group?P<0.01?.Therefore,the dose selected in this study included 0,25,50,100?g/mL,and the exposure time was set at24 h.With the increase of DKK-1 concentration,the cell survival rate showed a declining trend.When the dose of DKK-1 was 50 ng/mL,the cell survival rate was 93%,showing no statistical difference compared with the control group.2.PM_2.5.5 causes the increase of cell methylation level.Compared with the control group,DNMT1 protein,DNMT3b mRNA expression level and p16 gene methylation level increased with the increase of toxic dose,and the effect was strongest at the concentration of 100?g/mL?P<0.01?.3.PM_2.5.5 causes changes in cell EMT protein levels.Compared with the TGF-?1treatment group alone,Vimentin protein expression level was up-regulated and E-cadherin protein expression level was decreased as the dose increased?P<0.01?.4.PM_2.5.5 activates Wnt/?-catenin signal pathway.The expression levels of APC protein,APC mRNA and DKK-1 mRNA decreased with the increase of p-GSK-3?/GSK-3?ratio and expression levels of?-catenin and p-GSK-3?protein?P<0.01?.5.DKK-1 inhibits the Wnt/?-catenin signal pathway activated by PM_2.5.Compared with the single exposure group,the p-GSK-3?/GSK-3?ratio and the expression levels of?-catenin and p-GSK-3?protein decreased in the PM_2.5.5 and DKK-1 co-incubation groups,while the expression levels of APC protein and mRNA were up-regulated,both of which were statistically significant?P<0.01?.6.After DKK-1 inhibits PM_2.5.5 to activate Wnt/?-catenin signal pathway,p16methylation and cell EMT protein levels are changed.Compared with the single exposure group,the DNMT1 protein,DNMT3b mRNA expression level and p16methylation level were decreased in the PM_2.5.5 and DKK-1 co-incubation group,and the EMT protein Vimentin expression level was decreased while the E-cadherin protein expression was up-regulated?P<0.01?.ConclusionsThis study confirmed that atmospheric PM_2.5.5 could effectively induce methylation alteration of p16 tumor suppressor gene promoter,cell EMT and changes in tumor biological behavior in Beas-2B and A549 cells.The findings showed that atmospheric PM_2.5.5 plays an important role in the occurrence and development of lung cancer,and Wnt/?-catenin signaling pathway played an important regulatory role in the occurrence and development of lung cancer.This study proved that DKK-1 could be effectively rescued the methylation alteration of p16 tumor suppressor gene promoter caused by atmospheric PM_2.5.We will further explore the mechanism of DKK-1 regulating Wnt/?-catenin signaling pathway and the role of autophagy in methylation alteration of p16 gene and changes in tumor biological behavior induced by atmospheric PM_2.5.