The Role Of Circ＿010009 On PM_2.5-induced DNA Damage In Multiple Organs Of Male ICR Mice

Background and objectAmong atmospheric particulate matter（PM）,fine particulate matter(PM_2.5)with an aerodynamic diameter of less than _2.5μm is the main cause of global air pollution and poses a huge threat to human health.Toxicological and epidemiological studies have shown that PM_2.5 exposure can cause adverse effects on the respiratory system,cardiovascular system,urinary system,digestive system,reproductive system and even nervous system,resulting in damage to multiple organs such as lung,heart,liver,kidney,spleen,etc.The mechanism of PM_2.5-induced damage to health often involves immune toxicity,inflammatory response,oxidative stress,cell apoptosis and DNA damage.PM_2.5exposure can have serious genotoxic effects on animals,cells and even humans.DNA damage is considered to be a key event in PM_2.5-mediated genotoxicity.DNA damage is a more complex response,which is more likely to lead to instability and incompleteness of the genome,leading to cell senescence,cell death and even cancer.PM_2.5 exposure will increase the level of oxidative DNA damage of multiple organs in mice,but the pathological damage and mechanism of various organs after PM_2.5 exposure are not consistent.The mechanism of PM_2.5-induced DNA damage in multiple organs needs to be more comprehensively studied.Circular RNAs（circ RNAs）,a novel class of nc RNAs,are produced by covalent linking of 3’-5’phosphodiester bonds at the junction site.^{#1}circ RNAs are abundant,diverse,and highly conserved molecules with covalently closed loop structures in mammals,which exhibit tissue/developmental-stage-specific expression.According to the location of circular RNA in the cell,it can perform different functions.For example,circ RNAs in the cytoplasm can perform biological functions via binding micro RNAs（mi RNAs）and RNA-binding proteins（RBPs）,while circ RNAs in the the nucleus are involved in the regulation of gene transcription,alternative splicing and chromatin interactions.mi RNAs and long non-coding RNAs（lnc RNA）can play an important role in DNA damage after exposure to environmental chemicals.However,the function and mechanism of circ RNA in regulating DNA damage after exposure to environmental chemicals are still unclear.This study amid to explore the effects of PM_2.5 exposure on the pathological changes,DNA damage,DNA repair and circ RNAs expression of male ICR mice,focusing on the role and mechanism of circ RNAs in the DNA damage of multiple mouse organ after PM_2.5 exposure.MethodIn the process of DNA damage caused by PM_2.5,male ICR mice and RAW264.7cells were mainly selected as the research objects.Histopathological examination,western blotting and ELISA were used to detect organ damage and DNA damage in multiple mouse organ caused by PM_2.5.The cytotoxicity induced by PM_2.5 was detected by CCK8 and LDH assay.Western blotting alkaline comet assay and immunofluorescence analysis were performed to detect the degree of cellular DNA damage induced by PM_2.5.The influence of PM_2.5 on DNA repair pathway was explored by q RT-PCR and Western blotting.Through the high-throughput sequencing of circ RNA in mouse lung tissues,q RT-PCR selected and verified that PM_2.5 caused differential expression of circ RNA.The characteristics of circ RNA were confirmed by nucleoplasmic separation experiments and RNAse R treatment.With designing and synthesizing circ RNA interference sequences and overexpression vectors,gain-of and loss-of-function assays of circ RNA were carried out to explore the function of circ RNA in PM_2.5-induced DNA damage and DNA repair in RAW264.7 cells.We explored the correlation between circ RNA and DNA damage or DNA repair through Pearson correlation analysis.ResultsHistopathological examination of multiple mouse organ showed that PM_2.5exposure can cause alveolar capillary congestion,alveolar wall thickening,and a large number of neutrophil infiltration around the bronchus in lung;PM_2.5exposure can cause increased inflammatory cell infiltration around glomeruli and exfoliated epithelial cells in the lumen of the renal tubules in kidney;PM_2.5 exposure also induced blurred boundary between red pulp and white pulp,and more extramedullary hematopoietic cells and neutrophils in the red pulp.Western blotting showed that PM_2.5 exposure promoted phosphorylation of histone H2AX（γ-H2AX）in the lungs,kidneys and spleen of mice,indicating that PM_2.5 can cause DNA damage in the lungs,kidneys and spleen of mice.It was shown that PM_2.5 can promote the secretion of8-hydroxydeoxyguanosine（8-OHd G）by ELISA assay,which indicated that PM_2.5induced oxidative DNA damage in the blood of mice.A dose-dependent decrease in cell viability was observed after exposure to PM_2.5by CCK-8 assay,and a dose-dependent increase in LDH activity was observed in RAW264.7 cells exposed to PM_2.5 by LDH release assy.Western blotting showed that a dose-dependent increase in theγ-H2AX protein level in RAW264.7 cells exposed to PM_2.5by western blotting,alkaline comet assay showed that PM_2.5 could increase the Olive tail moment in RAW264.7 cells,and cellular immunofluorescence assay showed that PM_2.5 can increaseγ-H2AX protein level in RAW264.7 cells,which indicated that PM_2.5 can cause DNA damage in RAW264.7 cells.qRT-PCR showed that PM_2.5 can reduce the m RNA levels of Lig4 and Dclre1c in the lung,kidney,spleen and blood of mice,which indicated that PM_2.5 can inhibit non-homologous end-joining（NHEJ）repair.q RT-PCR and western blotting showed that Lig4 and Dclre1c were significantly downregulated at both the m RNA and protein levels in a dose-dependent manner in RAW264.7 cells exposed to PM_2.5.Through high-throughput sequencing of circ RNA in mouse lung tissues,it was found that PM_2.5 caused upregulation of circ＿010009 in lung,spleen,blood of mice as well as in RAW264.7 cells,and downregulation of circ＿010009 in kidney of mice.The stability of circ＿010009 was demonstrated by using RNase R,and showed that endogenous expression of circ＿010009 was resistant to excessive RNase R digestion while linear m RNAs were seriously degraded by RNase R treatment,which also supported that a natural circ RNA form of the Cabin1 gene existed inside.Then we investigated the localization of circ＿010009 in RAW264.7 cells using subcellular fractionation and localization assays,and we found that circ＿010009 was primarily localized to the cytoplasm of RAW264.7 cells.To investigate the role of circ＿010009 on PM_2.5-induced DNA damage,we used a loss of function method in RAW264.7 cells.Compared with the si-NC+PM_2.5exposure group,the level ofγ-H2AX protein in the si-circ＿010009+PM_2.5 group was significantly reduced,which showed that knockdown of circ＿010009 can reduce DNA damage.Next,we also investigated the function of circ＿010009 using a gain of function method.Compared with mock+PM_2.5 exposure group,theγ-H2AX protein level of OE-circ＿010009+PM_2.5 group increased significantly,which suggested that overexpression of circ＿010009 aggravated DNA damage.Importantly,the positive correlation between circ＿010009 expression and theγ-H2AX protein level were validated in lung and spleen,while negative correlation between circ＿010009expression andγ-H2AX protein level was verified in kidney via Pearson correlation analysis.Pearson correlation analysis also showed that circ＿010009 and 8-OHd G levels in blood were positively correlated.To investigate the role of circ＿010009 on PM_2.5-induced inhibition NHEJ repair,RAW264.7 cells were transfected with circ＿010009 interference sequence/overexpression plasmid.Compared with the si-NC+PM_2.5 exposure group,the m RNA and protein levels of Lig4 and Dclre1c in the si-circ＿010009+PM2.5group were significantly increased,which indicated knockdown of circ＿010009 can alleviate the inhibition of NHEJ repair caused by PM_2.5.On the contrary,compared with the mock+PM_2.5 group,the m RNA and protein levels of Lig4 and Dclre1c in the OE-circ＿010009+PM_2.5 group were significantly decreased,which implied that overexpression of circ＿010009 aggravated PM_2.5-induced inhibition of NHEJ repair.Pearson correlation analysis indicated that expression levels of circ＿010009 and Lig4or Dclre1c were negatively correlated in lung,spleen and blood,but positively correlated in kidney of mice.Conclusion1.PM_2.5 exposure can cause histopathological alterations and DNA damage in multiple organs of male ICR mice.2.PM2.5 exposure induces cellular toxicity and DNA damage in RAW264.7cells.3.PM_2.5 exposure can inhibit NHEJ repair in multiple organs of male ICR mice as well as in RAW264.7 cells.4.PM2.5 exposure induces upregulation of circ＿010009 in multiple organs of male ICR mice as well as in RAW264.7 cells,and circ＿010009 regulates the DNA damage induced by PM_2.5.5.circ＿010009 promotes PM_2.5-induced DNA damage via inhibition of NHEJ repair,and it is strongly correlated with NHEJ repair-related genes in multiple mouse organs,as well as in the blood.