The Function And Mechanism Of CircRNA005962 In The DNA Damage Of Respiratory Tract Epithelial Cells Induced By Carbon Black Nanoparticles

Background and purposeCarbon black refers to the product when the fuel is incompletely burned.It has been included in the 2B carcinogens organized by the International Agency for Research on Cancer.The toxic destructive force of carbon black particles in pharmacology is closely related to the size of their particles.Compared with the traditional micron-sized carbon black particles environmental chemicals,the cells induced by nano-sized carbon black particles are more severely damaged.Research evidence shows that carbon black nanoparticles can not only cause cytotoxicity and aggravate human respiratory diseases,but can even cause DNA chain damage.Circular RNA is a research hotspot of non-coding RNA.A large number of studies have shown that circular RNA plays an important regulatory function in the body’s health effects caused by chemical exposure.Therefore,exploring the role of circ RNA in the mechanism of DNA damage caused by carbon black nanoparticles can provide a more in-depth explanation and reveal the pathogenic mechanism caused by carbon black nanoparticles from an epigenetic level.Establish a model of DNA damage of airway epithelial cells caused by carbon black nanoparticles close to the actual exposure level,search for functional circ RNA and explore its molecular mechanism in carbon black nanoparticles-induced DNA damage of human airway epithelial cells.MethodTEM carbon black nano-particle characterization was used for identification.Based on the actual exposure threshold of particulate matter,articles related to particulate matter deposition in the lungs,and MPPD software model data,the exposure doses for in vitro cell models and in vivo animal models were determined.16 HBE,BEAS-2B cells and BALB/c mice were selected as the research objects.The model of DNA damage caused by carbon black nanoparticles was constructed in vitro,and the cell viability and oxidative stress level were detected by CCK-8,LDH and ROS experiments.ELISA,Western Blot,and neutral comet assay were used to detect DNA damage indicators such as 8-OHd G,γ-H2 AX,DNA double-strand breaks,etc.Western blot and q-PCR were used in in vivo experiments to evaluate the occurrence of DNA damage in the lungs of mice exposed to carbon black nanoparticles.Different circ RNAs were selected by high-throughput sequencing combined with q-PCR.Construct a research system for circ RNA interference and overexpression,combined with carbon black nanoparticle exposure,and perform experiments by flow cytometry,ELISA,Western Blot,immunofluorescence,neutral comet assay and other methods.It was detected that circ RNA005962 expression changes on human bronchial epithelial cells The cell cycle,ROS,8-OHd G andγ-H2 AX and other DNA damage indicators.The specific binding protein of circ RNA005962 was determined by RNA pulldown experiment,and the interaction between the three was verified by Western Blot and q-PCR under the combined exposure of carbon black nanoparticles.ResultCarbon black nanoparticles have a nanometer size in the solution,the DLS is258.84±3.34 nm,and the Zeta potential is-57.54±0.44.The results of in vitro experiments show that carbon black nanoparticles can inhibit cell viability,induce cell ROS levels and DNA oxidative damage,and the level of DNA damage caused by carbon black nanoparticles increases with the increase of the exposure concentration.The results of in vivo experiments show that carbon black nanoparticles can cause DNA damage in mice,whether in the low-exposure group or in the high-exposure group,and the level of DNA damage in the high-exposure group is more serious.circ RNA005962 showed high expression in the carbon black nanoparticles exposed group.After circ RNA005962 interference combined with carbon black nanoparticles were exposed,the DNA damage level was higher than that of the control group;overexpression circ RNA005962 combined with carbon black nanoparticles exposed,the DNA damage level was lower than the control group,indicating that circ RNA005962 in carbon black nanoparticles caused DNA damage in bronchial epithelial cells Play a regulatory role in the response.The RNA pulldown experiment proved that circ RNA005962 can directly bind to the RNA binding protein FUS and is positively regulated by FUS;circ RNA005962 can bind to the DNA repair protein LIG4.After interference and overexpression of circ RNA005962 and exposure to carbon black nanoparticles,circ RNA005962 can reversely regulate the level of LIG4,further confirming that circ RNA005962 plays a regulatory function in the process of DNA damage.Conclusion1.CBNPs can cause DNA damage in human bronchial epithelial cells and mouse lungs.In the process,circ RNA005962 functions as a repair-promoting gene.2.In the process of CBNPs-induced DNA damage in human bronchial epithelial cells,circ RNA005962 directly binds to FUS and is directly regulated by it;at the same time,circ RNA005962 directly binds to LIG4 to positively regulate its expression,thereby regulating DNA damage.