Mechanism Of G2/M Phase Arrest In A549 Cells Induced By PM_2.5 Dust-fall Collected From Different Area

Objective:As is known to all,the sources of atmospheric fine particulate matter(PM_2.5)mainly include industrial waste gas and vehicle exhaust emissions,coal burning and construction dust.And PM_2.5 is a signature pollutant for evaluating air pollution.In recent years,with the rapid development of China's industrial economy,environmental problems caused by PM_2.5 are becoming increasingly prominent,And air pollution has become the fourth risk factor that seriously endangers public health.PM_2.5 enter into the body through breathing,it is closely related to respiratory system diseases.Therefore,the toxic effects and mechanisms of PM_2.5 have gradually become the focus of many scholars at home and abroad.In this experiment,we choose the PM_2.5 dust-falls in four different regions of Ningxia Yongning?YN?,Qinghai Xining?XN?,Hebei Shijiazhuang?SJZ?and Sichuan Mianyang?MY?as research objects,the A549 cells cultured in vitro are used as test cells.Then the A549 cells were acute exposed to PM_2.5 dust-falls collected from different areas to establish a cell model.On this basis,the toxicity effects and cycle change of PM_2.5 dust-falls reduction on A549 cells in four different regions were analyzed,and its possible toxic mechanism were explored.It is of solid foundation to promote the development of environmental health related research in China.Methods:?1?The particle size dispersion in four different regions were determined by the laser particle size analyzer after grinding.The dust samples were made into dust pellets,and the main phases were analyzed by XRD.?2?Select A549 cells of exponential growth stage and growth in good condition.A549 cells were incubated in 96-well plates,and treated with 200?L different concentrations including 0?control group?,12.5,25,50,100,and 200?g/mL of PM_2.5 dust-falls for 12,24,and 48 h,respectively.And the blank control group and the dust control group were set.Then the MTT method was used to detect the survival rate of A549 cells,and analyze the correlation between survival rate and dust concentration.?3?The A549 cells were inoculated into the 12-well plates of the built-in sterilization climbing tablets,and added 100?g/mL PM_2.5precipitation suspension.After 24 hours of co-culture,the climbing tablets were taken out and the W-G dye was staining after fixation.Then the cell morphology was observed in the oil microscope.?4?A549 cells were inoculated in 12-well plates and added 0,12.5,25,50,100 and 200?g/mL PM_2.5suspension.At the same time,A549 cells were inoculated in confocal culture dish and added 100?g/mL PM_2.5 suspension.All of them were infected for 24 h and the fluorescent probe DCFH-DA were used to labeled cells.The mean fluorescence intensity was detected by spectrofluorometer,and the images were analyzed by laser scanning confocal microscope.Subsequently,the correlation between ROS relative level and cell viability was analyzed.?5?A549 cells and100?g/mL PM_2.5 suspension were co-cultured in 6-well plates for 24 h,and Set up control group at the same time.Then the infected A549 cells were collected,and the cell cycle changes were detected by flow cytometry after staining with propidium iodide.?6?Based in the process of PM_2.5 dust exposure induced A549 cell cycle arrest,different regional PM_2.5 dust-falls exposed to A549 cell in the concentration of 100?g/mL.The expression level of cell cycle arrest related gene p53,p21,CDK1,c-myc and lncRNA H19 were detected by RT-PCR,and the expression of cycle protein Cyclin B was measured using Western blot test.?7?On the basis of the early stage of the infected model,through transfected H19 siRNA to disturb the expression of H19,and RT-PCR test was used to detect the expression of p53,c-myc and CDK1.Thereby to investigate the interaction between lncRNA and mRNA in the process of PM_2.5induced cell cycle arrest.And lay a solid foundation for study on the mechanism of PM_2.5 induced cell cycle arrest.Results:?1?The dust particles were dispersed by pure water,and most of the particle sizes were less than 2.5?m.The D90 values of dust particles in YN,XN,SJZ and MY were 2.011,2.208,2.423 and 2.397?m.In addition,except for the main phase quartz in the PM_2.5 dust-fall,XN and SJZ groups mainly doped with calcite,albite and dolomite,YN group mainly doped with albite and dolomite,and the MY group mainly doped with calcite.?2?Compared with the control group,when the concentration of PM_2.5 increased from 12.5?g/mL to200?g/mL,cell survival rate decreased with the exposure concentration increased at the same treatment time.In addition,in maintaining the concentration under the same conditions,the cell viability showed a decreasing trend with the increasing exposure time.It showing a good dose-time-effect relationship.?3?Compared with the control group,different regions of PM_2.5dust effect,cell death and cell debris suspension more fluid in cell culture,cell surface adsorption dust exposure group more fine particles.Part of the cell contour was disappeared,cell membrane was dissolved,cell micronucleus and eroded hole damage was appeared.?4?Compared with the control group,the intracellular ROS levels of A549 cells in YN,XN,SJZ and MY groups showed an increasing trend with the increase of PM_2.5 concentration.After the four groups of PM_2.5 dust reduction,the cell was damaged in different degrees,and the fluorescence intensity of the damaged cells increased significantly.In addition,there was a significant correlation between the level of intracellular ROS and cell vitality in YN,SJZ and MY groups.?5?Compared with the control group,after treated A549 cells with the four regional PM_2.5,there was no obvious change in S phase cells,and cells in G0/G1 phase decreased,cells in G2/M phase ratio was significantly increased.It was suggested that the infectious A549 cells stay in G2/M cells was prolong for a longer time to inhibit cell proliferation.?6?Compared with the control group,exposed to PM_2.5 in the YN,XN,SJZ and MY regions could increased the expression of G2/M phase relate genes p53,p21,c-myc and lnc RNA H19,and the expression of CDK1and cyclin B1 were decreased.?7?After transfection of H19 siRNA,the expression of H19 was successfully interfered,and the expression of p53,c-myc and CDK1 gene was further decreased.Among them,the inhibition of gene expression in XN group was the most significant.It was suggested that lncRNA H19 and c-myc can play a role in co-amplification,and participate in cell cycle progression through p53 binding,and interfere with low expression of H19 induce G2/M phase arrest more obviously.Conclusion:?1?PM_2.5 dust-falls in different areas treated on A549 cells can destroy cell morphology and inhibit cell proliferation.?2?PM_2.5 dust-falls in different regions treated on A549 cells can increase the ROS production,then induce intracellular oxidative damage and interfere cell cycle progress.?3?Exposed to PM_2.5 dust-falls in different areas can inhibit the expression of CDK1 and cyclin B1 by activating p53 and p21 activity,and induced G2/M phase arrestin A549 cells.?4?After short-term exposure to PM_2.5 dust-falls in different regions,lncRNA H19 may played a specific role in oncogene in A549cells,and it participated in cell cycle progression by binding to p53 and c-myc.