Application Of Rna Interference In The Study Of The Activation And DNA Damage Of Benzo (a) Pyrene Mediated By5-lipoxygenase In Human Bronchial Epithelial Cell

[Objectives] The relationship between the oxidation of benzo(a)pyrene and the DNA oxidative damage in HBE cells were studied to confirm that lipoxygenase(LOX) is an alternative pathway of the oxidation of xenobiotics mediated by cytochrome P450and provide strong evidence that LOX siRNA probably prevent the toxicity of chemicals by inhibit the activity of LOX.[Methods] Gene silence experiment:pAJ-U6-shRNA-CMV-GFP recombinant is formed by means of cutting the plasmid with the Agel and EcoRI enzyme and jointing the T4Ligase and shRNA Oligo fragments. Then It transfects293T cell through lentivirus vector to obtain the virus concentrated solution which is used to infect the target cell. The PCR and Western-blot methods are employed to detect the effect of gene silence.Cellular experiment:After HBE cells and cells disposed with5-LOXsiRNA exposure to different concentrations of benzo(a)pyrene. The effect of benzo(a)-pyrene on cell survival rate were assessed by reductions of tetrazolium dye(MTT),the level of protein expression of5-lipoxygenase in the cells is tested by western-blot, and the DNA damage by the single cell gel electrophoresis and flow cytometry. At last, the effect of cells treated by5-LOXsiRNA and chemical inhibitor after exposure to benzo(a)pyrene on cell toxicity and DNA damage in the cells are detected. The differences between5-LOXsiRNA and chemical inhibitor cells are compared.[Results] Real-time PCR and Western-blot results prove that the effect of5-LOX gene locus silence is successful after sequencing the gene only after identifying transformer and positive clones and infecting HBE cells. Benzo(a)pyrene can induce5-lipoxygenase protein expression, but AA-861can not in HBE. Benzo(a)pyrene causes significant toxic action and DNA damage in HBE, which can be inhibited by5-LOXsiRNA and specific inhibitor of5-LOX (AA-861), with the former more remarkable.[Conclusions]5-LOX protein expression in HBE can be regulated by benzo (a) pyrene. The specific inhibitor of5-LOX (AA-861) and5-LOXsiRNA can suppress the cytotoxicity and DNA damage caused by benzo(a)pyrene. It is infered that the oxidation of benzo(a)pyrene by5-LOX turns into electrophiles that covalently bind to DNA and induce DNA damage, which is involved in the carcinogenicity of benzo(a)pyrene and can be effctively restrainted by means of RNA interference.