E2F1-invovled Mechanism Of Pulmonary Injury Induced By Diesel Exhaust Particle Exposure

PM2.5 is the primary pollutant of air pollution in China;it can be deposited in the depths of lung,through the blood circulation into organs,resulting in more serious health effects.Diesel exhaust particles(DEPs)are the largest single source of PM2.5 from motor vehicle exhaust.In 2013,the World Health Organization(WHO)defined DEP as a class I carcinogen.Exposure to DEP is associated with the development of respiratory disease and lung cancer.Despite the extensive research about the effects of DEP on human health,the underlying molecular mechanisms of DEP and respiratory disease remain unclear.E2F transcription factor family is an important regulatory protein in cells;E2F1 can activate DNA synthesis and induce quiescent cells into the S phase,which is the key to apoptosis,immune response and chronic inflammation.Therefore,we investigated the regulation of target gene expression in human alveolar epithelial cells,and explored the molecular mechanism of E2F1 in the pulmonary injury induced by DEP.This study can provide clues for the mechanisms of cytotoxicity induced by DEP.1.Effects of DEP exposure on transcriptomic expression profile of lung tissue cells1.Transcriptomic expression profile was detected to analyze the effects of DEP on mRNA and protein expression profiling of A549 cells after exposed to 50 ?g/mL DEP for 24 h.To analyze the data of microarray profile,first of all,we screened differentially expressed genes(DEGs)after DEP treatment.The results showed that a total of 8000 genes were differentially expressed at mRNA level,among which 2512 genes were up-regulated and 5488 genes were down-regulated(FC?2 and P<0,05).There were 254 differentially expressed genes at protein level,167 genes were up-regulated and 87 genes were down-regulated(FC>2 and P<0.05).As for the same trend in mRNA and protein levels,55 genes were selected,14 of them were up-regulated and 41 were down-regulated(FC?2 and P<0.05).Combined with transcription factor-target regulatory network analysis,top5 transcription factors FLI1,HNF4A,GABP,XPN2 and E2F1 were selected according to the enrichment number of regulated genes,among which 14 target genes(NXT1,DDX46,PPIG,DDX17,NUDT21,NXF1,DKC1,MBNL1,YTHDC1,PABPNI,UPF3B,PIN4,NONO and FILIP1)were regulated by the transcription factor E2F1.The Gene Ontology(GO)analysis was conducted to enrich and classify DEGs functionally by Database for Annotation,Visualization and Integrated Discovery(DAVID)6.7 Online Gene Annotation System.GO analysis showed that E2F1 regulates 14 target genes involved in RNA processing,RNA splicing,mRNA processing,mRNA metabolism process and RNA transport process.2.A549 cells were exposed to DEP(0,50,100 ?mL)for 24 h,qRT-PCR was performed to verify the results of the microarray.The trends of down-regulation of PPIG,DDX17,NUDT21,NXF1,DKC1,MBNL1,PABPN1,NONO and FILIP1 genes in qRT-PCR assay were similar to those in the results of mRNA microarray analysis,which proved that the gene expression chip was reliable.Therefore,DEP exposure can cause changes in the expression profile of A549 cells and downregulate the expression of E2F1-regulated genes,suggested that E2F1 may play an important role in DEP-induced lung injury.2.E2F1 regulates the cytotoxicity induced by DEP in lung tissue cellsTo investigate the role of E2F1 in the lung injury induced by DEP,we first studied the cytotoxicity of DEP on A549 cells and the mechanism of E2F1 at the cellular level.1.Toxic effects of DEP on A549 cells1.1 The cell viability of A549 cells was measured by CCK-8 colorimetric assay at lday,2day,3day,4day,5day,6day,7day after exposure to DEP(0,12.5,25,50,100,125 ?g/mL).The results showed that the cell proliferation activity was significantly decreased after exposure to DEP(P<0.05),moreover there was a dose-response relationship.1.2 The morphological of A549 cells exposed to DEP for 24h.1.2.1 A549 cells were exposed to 0,12.5,25,50,100[ig/mL DEP for 24 h.The number of adherent cells decreased,cells appear to be shrunk with pseudopodia disappeared.A large number of DEP particles were observed under the microscope.The number of death cells increased with the exposure dose.1.2.2 A549 cells were exposed to 50,100 ?g/mL DEP for 24 h and observed under transmission electron microscopy.The uptake of massive particles could be observed in the cytoplasm scope,while in the high-dose DEP-treated A549 cells,a large number of swollen mitochondria were observed in the cytoplasm.1.3 A549 cells were exposed to 0,12.5,25,50,100 pg/mL DEP for 24 h.The effect of DEP on apoptosis and cell cycle of A549 cells was detected by flow cytometry.The current results showed that the cell apoptosis rate increased with the concentration of DEP,and the cell death rate of A549 cells was significantly increased after 100 ?g/mL DEP exposure(P<0.05).Cell cycle results showed that the cells were mainly arrested in S phase and G2/M phase(P<0.05).2.E2F1 regulates the cytotoxic effect of DEP on A549 cells2.1 pcDNA-3.1-empty plasmid and pcDNA-3.1-His-E2F1 plasmid were transfected into A549 cells for 48 h.QRT-PCR and Western Blot were used to detect the mRNA and protein expression of E2F1 in A549 cells.mRNA and protein of expression levels of E2F1 were significantly increased(P<0.05)after E2F1 plasmid transfection compared with transfected empty plasmid group.The results showed that E2F1 overexpressing A549 cell line was successfully constructed.2.2 E2F1 overexpression A549 cells exposed to 50 ?Rg/mL DEP for 24 h.The effect of E2F1 on the apoptosis and cell cycle of A549 cells induced by DEP was detected by flow cytometry.The results showed that overexpression of E2F1 could significantly reduce the apoptosis and mortality of A549 cells(P<0.05),and rescue the S arrest of A549 cells following DEP exposure(P<0.05).2.3 The mRNA and protein expression of PPIG,DDX17,NUDT21,NXF1,DKC1,MBNL1,PABPN1,NONO and FILIPI were measured by qRT-PCR and Western Blot after exposed to DEP.We found that E2F1 overexpression A549 cells exposed to 0,25,50 ?g/mL of DEP for 24 h,the mRNA levels of NUDT21,PABPN1,PIN4,NXF1 and NXT1 were significantly increased(P<0.05)compared with the control group.Western Blot results showed that PABPN1 protein expression was significantly reduced,and there was dose-response relationship.E2F1 overexpressed A549 cells were exposed to 0,25,50 ?g/mL DEP for 24 h,PABPN1 protein expression level was rescued to normal levels compared with the control group.The results showed that DEP could induce the down-regulation of mRNA and protein expression of E2F1-related target genes in A549 cells,changing the apoptosis and cell cycle biological processes and affecting the proliferation of A549 cells.Overexpression of E2F1 reduced the mRNA and protein expression levels of DEP-related genes,partially restored to normal levels,reducing the apoptosis and alleviating cell cycle arrest of A549 cells induced by DEP.Suggested that E2F1 may play an important regulatory role in the process of cytotoxicity induced by DEP exposure in lung tissue cells.3.Construction of E2F1Tg/Tg transgenic mouse modelConstruct the overexpression E2F1Tg/Tg transgenic mouse model,establish DEP tracheal drip exposure mouse model.1.The construction of E2F1Tg/Tg transgenic mouse model E2F1Tg/Tg transgenic mice were constructed by Guangzhou Saiye Biotechnology Co.,Ltd.according to Tet-on system.The transgenic mice were transfected into the tissue-specific expression vectors Cre,E2F1 and Tet3G-carrying vector plasmids and need to be identified before experiments.The positive transgenic mice were identified when Cre,E2F1 and Tet3G were expressed as positive.2.Establish a model of endotracheal instillation in E2F1Tg/Tg transgenic mice,doxycycline feeding(2 mg/mL)were given to induce E2F1 overexpression in transgenic mice.The transgenic mice were divided into E2F1Tg/Tg control group,E2F1Tg/Tg&DEP exposure group,E2F1+/+ control group and E2F1+/+&DEP group.4.In vivo toxicity caused by DEP and the role played by E2F1According to the model of transgenic mice,the toxicities of DEP exposure and the regulation of E2F1 were investigated.1.Toxicity of endotracheal instillation with DEP in vivo1.1 The histopathological changes of the mice were observed by histopathological observation and routine pathological staining.DEP was deposited in lung tissue after DEP exposure;compared with E2F1+/+&DEP group,E2F1Tg/Tg&DEP mice had lung lobe atrophy and gray-white vegetation;histopathological examination of pulmonary tissue revealed that pathological changes of the lungs were observed,including alveolar hyperemia,thickening of alveolar wall,atypical hyperplasia of lung tissue,consolidation of surrounding alveoli,and infiltration of inflammatory cells;the lung injury showed a significant increase in the scores of the DEP exposure group compared with the E2F1Tg/Tg control group,the score of E2F1+/+&DEP mice was lower than that of E2F1Tg/Tg&DEP exposure group;HE staining showed that the hepatic tissue of mice exposed to DEP increased the volume of liver cells,the structure of liver cords disappeared,cytoplasm was loose with the nucleolysis occurred,at the same time,infiltration of inflammatory cells in the hepatic sinus and inflammatory cells in the hepatic lobules focal accumulation were observed.1.2 The expression of E2F1-related genes in pulmonary tissues of mice exposed to DEP was detected by qRT-PCR and immunohistochemical staining.The results showed that the mRNA expression level of E2F1-related genes in DEP mice model was changed.Compared with the control group,the mRNA expression of PABPN1,NXF1,PIN4,DDX17 in lung tissue of E2F1Tg/Tg&DEP group was significantly decreased(P<0.05);The mRNA expression of PABPN1,NXF1,PIN4,DDX1,in the E2F1+/+&DEP mice were partially restored to normal levels with the increase of transcription factor E2F1 expression.Immunohistochemistry results showed that the positive cells of E2F1 and PABPN1 in the lung tissue of DEP-treated mice were significantly decreased(P<0.05),while the positive cells of PANPN1 in E2F1+/+mouse lung tissue were significantly increased compared with E2FTg/Tg.In conclusion,the endothelium-deprivation of DEP could lead to the damage of organs and pathologic changes in mice.The expression of related genes in lung tissue was down-regulated,and the transcription factor E2F1 could regulate the down-regulated genes.Suggested that transcription factor E2F1 plays an important role in the process of lung injury in mice after exposed to DEP.