Screening Of Paraquat Resisitence Related Genes Based On CRISPR/Cas9 Genome-wide Library

Background: Paraquat is one of the most widely used herbicides in the world.Its chemical name is 1-1-dimethyl-4-4-bipyridine cationic salt.Because of accidental ingestion or suicide,paraquat is extremely harmful to human beings.The death rate after poisoning is as high as 80% to 90%.Currently,there is no specific antidote.The toxicity of paraquat mainly comes from its oxidative property.After entering the human body,it will damage cell mitochondria,lipid,DNA molecules and proteins by producing a series of reactive oxygen species,resulting in cell death.However,inhibition of reactive oxygen species or the use of antioxidants cannot effectively remove the toxicity of paraquat.According to existing studies,oxidative stress,inflammatory response,complement activation,fibrosis and autophagy of cells are all involved in the cell death caused by paraquat,and the molecular mechanism is very complex,which is still not fully understood,and resistant drugs or inhibitors to treat paraquat poisoning has little effect.Objective: we used CRISPR/Cas9 in human lung cancer epithelial cells A549 to screen potential anti-paraquat genes,and the further study of the function and mechanism of the screened genes will provide theoretical basis or ideas for the treatment and molecular mechanisms of paraquat poisoning.Methods: The CRISPR/Cas9 genome-wide knockout amplification and packing slow virus library plasmid,test the virus infected after drop degree of A549 cells control MOI(0.3)or less,after puro screening,using paraquat treatment with 10 generations,the whole genome sequencing and bioinformatics analysis,enrichment sg RNA corresponding candidate genes was enmerged after eliminating false positive and false negative results.The candidate gene knockout sg RNA was designed and packaged with lentivirus to infect A549 cells to establish the candidate gene knockout cell line.CCK8 survival assay was used to detect the resistance of candidate knockout genes to paraquat induced death.In order to study the molecular mechanism of tolerance of candidate genes to paraquat toxicity,q PCR and Western blot were used to test whether mir-6766 was involved in regulating the expression of POR.The expression level of HMGB1 in the supernatant of paraquat treated cells was detected by ELASA,and the binding effect of HMGB1 and paraquat was verified by the Cellular Thermal Shift Assay and surface plasmonic resonance experiments.The differentially expressed genes in HMGB1 knockout cells were analyzed by RNA-seq technique,and knockout cell lines with related differentially expressed genes were constructed to verify the resistance of HMGB1 to paraquat.The transcription,translation and secretory expression levels of IL-11 under paraquat treatment were detected by q PCR,Western blot and ELASA.The expression levels of IL-11 and associated collagen in human recombinant HMGB1 were detected by q PCR.To detect the protective effect of glycyrrhizic acid,an HMGB1 inhibitor was used.Results: By using the CRISPR/Cas9 human genome knockout library in A549 cells,the potential anti-paraquat genes enriched with sg RNA were obtained.Through the identification of potential candidate genes,it was found that POR,mir-6766 and HMGB1 knockout celllines were resistant to paraquat induced cell death,and glycyrrhizin,an inhibitor of HMGB1,could protect cells from paraquat poision.It was found that the HMGB1 protein secretion level increased after treatment with different concentrations of paraquat.Some ECM components of HMGB1 knockout cells decreased,and cell migration ability increased after knockout of HMGB1 gene.IL-11 knockdown cells showed a paraquat tolerance phenotype.The protein and secretion levels of IL-11 were increased in A549 cells treated with paraquat.When human HMGB1 recombinant protein was used to stimulate A549 cells,the transcription levels of COL1A1 and COL1A2 were increased,the ratio of MMP2/ TIMP1 m RNA was unbalanced,and the secretion level of IL-11 was increased.Conclusions: In this study,the CRISPR/Cas9 human genome knocko ut library was used to successfully screen out POR,HMGB1,mir-6766 an d other potential paraquat-resistence genes.The molecular mechanism of paraquat tolerance mediated by these candidate genes was preliminarily e-xplored.It was found that mir-6766 might mediate the paraquat tolera-nce phenotype by regulating the expression of POR.HMGB1 plays an important role in paraquat induced cell death and may affect the progres sion of pulmonary fibrosis by further stimulating the secretion of the pulmonary fibrosis factor IL-11.These works provide theoretical basis for searc-hing for antidote methods of paraquat.