The Mechanism Research Of Black Carbon Promoting The Proliferation Of Lung Epithelial Cells Through DNA Methylation

Objective:The problem of air pollution has become increasingly serious.In recent years,large-scale and high-intensity haze weather has continued to occur frequently throughout the country.Atmospheric fine particulate matter(PM2.5)has become an important environmental health risk factor.In recent years,the increasing pollution of atmospheric fine particles is closely related to the incidence of malignant tumors such as heart disease,chronic obstructive pulmonary disease,stroke and lung cancer.Black Carbon(BC)is the main component of atmospheric fine particles,and it plays an important role in the air pollution and the toxic effects of atmospheric fine particles in my country.DNA methylation is an important part of epigenetics.Studies have shown that exposure of carbon nanomaterials can cause abnormalities in a variety of cellular processes and signaling pathways by causing changes in DNA methylation.Other studies have found that exposure to fine particulate matter in the atmosphere can affect the genomic DNA methylation level in the blood of a population.It can be seen that atmospheric fine particles can change the level of DNA methylation and promote cell deterioration.However,there is no report to systematically explore the effect and mechanism of black carbon on the level of genomic DNA methylation.This article uses a variety of DNA methylation detection and sequencing analysis methods to explore the effects of black carbon on the proliferation of normal and tumor cells,and in-depth reveals the molecular mechanism of black carbon that promotes malignant cell proliferation through changes in DNA methylation,in order to effectively evaluate atmospheric fine particles.The health risks and toxicity mechanisms of toxic components provide a scientific basis.Methods1.Detect the characteristics of the three black carbon materials Printex U,SA4 B and C824455 through projection electron microscope technology,atomic force microscope technology,particle size and Zeta potential,analyze the dispersibility and structural stability of black carbon particles,and pass the particle size And electric potential to evaluate the performance of the material.2.The mouse lung tissue was stained and analyzed by HE staining to determine the damage effect of black carbon particles on mouse lung tissue;the levels of 5mc and 5hmc in mouse lung tissue were detected by immunohistochemistry,and the level of 5mc and 5hmc in mouse lung tissue was detected by Dot blot.The expression of5 mc and 5mc of DNA in mouse lung tissue was verified by immunohistochemical results to determine the effect of Printex U,SA4 B and C8224455 on the DNA methylation level of mouse lung tissue;and we used Western Blot technology to detect mice The protein levels of DNA methyltransferases DNMT1,DNMT3 A and DNA demethylases TET1 and TET2 in lung tissue reflect the process of black carbon specifically changing the level of DNA methylation.3.Screen the proliferation-promoting concentration of Printex U and SA4 B on lung cancer cell line A549 and lung normal epithelial cell line BEAS-2B through MTT experiment,and verify each other with EDU experiment;continue to use the selected black carbon concentration to promote proliferation under exposure Scratch test and Transwell test were used to detect the effects of Printex U and SA4 B on the migration and invasion of A549 cells;the selected black carbon concentration that promotes proliferation was used to detect the effects of Printex U and SA4 B on A549 by flow cytometry.The effect of cell and BEAS-2B cell cycle;after exposure at the same concentration,the expression of 5mc,5hmc,DNA methyltransferase and DNA demethylase in A549 cells and BEAS-2B cells was detected by Dot blot and Western Blot.4.Use bioinformatics technology to perform whole-genome methylation sequencing(Me DIP-seq)on mouse tissue and cell DNA samples,perform RNAseq sequencing on cellular RNA samples,obtain differentially expressed genes between the experimental group and the control group,and apply biological information The scientific method uses the two parts of Me DIP-seq and RNA-seq data to perform correlation analysis,and through gene function analysis,pathway analysis,cluster analysis,etc.,combined with literature search to screen out the differentially methylated and differentially expressed genes related to cell malignant proliferation and verify it.Result1.Compared with C824455,Printex U and SA4 B have stable structure,better dispersion and biocompatibility.2.C824455,Printex U and SA4 B in vivo exposure caused lung tissue damage and congestion in mice,and Printex U and SA4 B enhanced the level of DNA methylation in the lung tissue of mice.3.Exposure to low concentrations of Printex U and SA4 B promotes the proliferation and cycle of A549 cells and BEAS cells,enhances the level of oxidative stress,and Printex U also promotes the migration and invasion of A549 cells.4.Printex U and SA4 B enhanced the DNA methylation level of A549 cells and BEAS cells and changed the protein expression levels of DNMT enzyme and TET enzyme under proliferation-promoting concentration exposure.5.Exposure to Printex U and SA4 B caused differential methylation changes in genes related to cell trafficking and catabolism.ConclusionThe two black carbon particles,Printex U and SA4 B,promote the proliferation and cycle of normal lung epithelial cells BEAS-2B and lung cancer cells A549 through DNA methylation,and enhance the invasion and migration of A549 cells,and cause cell transport and catabolism.Differential methylation changes of genes in related pathways,but the specific molecular mechanisms are still being studied.Continue to explore the mechanisms in this way to provide a scientific basis for the effective evaluation of the health risks and toxicity mechanisms of the toxic components of atmospheric fine particles.