The Role And Mechanism Of PARP-1 Defect In The Carcinogenesis Of Benzopyrene

ObjectivePoly ?adenylate diphosphate ribose?multimers can be used in the presence of poly ?adenylate diphosphate ribose?transferase-1 ?poly ?ADP-ribose? polymerase-1,PARP-1??ADP-ribose? glycohydrolase,PARG),this reversible ADP-ribosylated modified protein acts on DNA repair ?poly?ADP-ribose? glycohydrolase,PARG? And apoptosis plays an important role.Our early studies have shown that benzo [a] pyrene?Ba?may induce epidermal growth factor receptor?EGFR?and ubiquitin carboxy terminal hydrolase L1 ?EGFR? during malignant transformation of cells?Ubiquitin C-terminalhydrolase L1,UCH-L1?was up-regulated in human bronchial epithelial cells?16HBE cells?and down-regulated in PARG-deficient cells.However,the corresponding effect was also found in PARP-1 deficiency.To investigate the expression of EGFR and UCH-L1 protein in the carcinogenesis of benzopyrene induced by BaP inhalation,and to investigate the effect of PARP-1 gene on BaP-induced carcinogenesis in BaP-induced lung injury And its possible mechanism,so as to provide the basis for the prevention and treatment of chemical carcinogenesis.Method1.Effects of PARP-1 Defect on BaP-induced Lung Cancer in Mice?1?Reproduce a sufficient number of PARP-1-deficient mice to identify PARP-1^-/-mice for exposure experiments;?2?WT mice and PARP-1^-/-mice were randomly divided into 8 groups:WT female mice control group,WT female mice exposure group,WT Male mice control group,WT male mice exposure group,PARP-1^-/-female mice control group,PARP-1^-/-female mice exposure group,PARP-1-/-male mice control group And PARP-1^-/-male mice were exposed to mice for 6h/day,5 days/week for 6 months.The survival status of mice was observed and recorded;?3?The content of Ba P in external exposure was measured by activated carbon tube method to monitor the concentration of exposure.;?4?The content of BDPE DNA adduct in whole blood was determined by competitive enzyme-linked immunosorbent assay?ELISA?to detect the effect of BaP in the body.?5?The lung cancer model was successfully established by lung histopathology.2.Analysis of the mechanism of PARP-1 in BaP-induced lung cancer in mice EGFR and UCH-L1 were used as the two indexes to detect the tumor.The expression of EGFR and UCH-L1 gene in WT and PARP^-/-mice were detected by real-time fluorescence quantitative PCR.Immunofluorescence and Western blot were used to detect the expression of EGFR and UCH-WT mice and PARP^-/-mice in order to analyze the mechanism of PARP-1 deficiency in BaP carcinogenesis.Result1.Effects of PARP-1 Defect on BaP-induced Lung Cancer in Mice?1?The mouse tail tissue DNA was extracted and PARP^-/-mice were identified by PCR;?2?The external exposure dose was?12.794±0.518??g/m³ during the6 months of BaP exposure;?3?The concentration of BPDE DNA in whole blood was detected by ELISA kit,suggesting that BPDE DNA is an indicator of BaP toxicity exposure.WT mice and PARP-1^-/-mice were treated with BPDE-DNA?p<0.05?.The concentration of BPDE DNA adduct in WT female mice was greater than that in PARP-1^-/-female mice?p<0.05?,and the difference was statistically significant?p<0.05?.The concentration of BPDE-DNA adduct in WT male mice was not significantly different from that of PARP-1^-/-male mice.The difference of BPDE DNA adducts between WT and PARP-1^-/-?p<0.05?,and the difference was statistically significant?p<0.05?;?4?Pathological observation of lung tissue found that WT female mice lung tissue in the diffuse alveolar septum thickening,alveolar wall destruction and alveolar fusion,visible nodules in lung tissue,pathological analysis of undifferentiated cell carcinoma,suggesting WT female mice lung cancer model was successfully established;WT male mice and PARP-1^-/-mice only diffuse alveolar septal thickening,alveolar wall destruction and alveolar fusion,no nodules,suggesting that WT male mice and PARP-1^-/-mice lung tissue did not form lung cancer.2.Analysis of the mechanism of PARP-1 in BaP-induced lung cancer in mice Real-time fluorescence quantitative PCR,immunofluorescence and western blot showed that the expression of EGFR protein in the lung tissue of BaD mice was higher than that of the control group?p<0.05?.The expression of UCH-L1protein in female mice was significantly different?p<0.05?,but the expression of UCH-L1 protein in male mice was not statistically significant.In PARP-1^-/-mice lung tissue?p<0.05?.The expression of UCH-L1 protein in female mice was lower than that in control group?p<0.05?,and the difference was statistically significant?p<0.05?The expression of UCH-L1 protein in male mice was not statistically significant.Conclusion?1?PARP-1 deficiency has the effect of inhibiting the occurrence of lung cancer in mice during the long-term exposure of BaP;?2?PARP-1 deficiency can regulate the expression of EGFR and UCH-L1 protein during BaP carcinogenesis.ADP ribosylation may regulate EGFR signaling pathway and ubiquitination process.