Circ₀001839 Regulates MYCL Mutation Frequency To Promote The Development Of Benzo（a）pyrene-induced Lung Cancer

Background and objectAs the main representative of polycyclic aromatic hydrocarbons(PAHs),benzo(a)pyrene(B[a]P)is a common environmental chemical in daily life,and there is increasing evidence to confirm the strong mutagenicity and carcinogenicity of B[a]P.Circular RNAs(circRNAs)can regulate genetic damage in the organism through DNA damage repair pathways,are an important bridge between epigenetics and genetics,and have great potential as biomarkers for early cancer exposure.There are more studies on the functional role of circRNAs in DNA damage,but the relationship between circRNAs and gene mutations remains unclear.This study focused on the relationship between circRNAs and gene mutations in human bronchial epithelial lung cancer cells induced by the environmental pollutant B[a]P during its development,which is important and innovative to reveal the toxicological effects and biological pathways of B[a]P on human health.Method1.Soft agar clone formation assay and plate cloning assay were performed to verify the malignant phenotype of 16HBE-T.2.Whole transcriptome sequencing was used to screen differentially expressed circRNAs,and q RT-PCR was used to validate the sequencing results.3.Agarose gel electrophoresis experiments were used to verify the loop-forming properties of circRNAs;FISH experiments and cytoplasmic cytosolic separation PCR were used to identify the localization of circRNAs in cells.Lentiviral vector transfection,Ed U and scratch experiments were used for the functional study of circRNA.4.Whole genome sequencing is used to detect mutated oncogenes and validated by sanger sequencing.5.Pyrophosphate sequencing was used to detect the expression frequency of different bases at the mutated sites.6.Statistical analysis software SPSS was used to organize and analyze the data.p-values less than 0.05 were considered to be statistically different.Result1.Malignantly transformed cells 16HBE-T had enhanced proliferative capacity and monoclonal formation,and possessed the characteristics of malignantly transformed cells.2.Circ＿0001839 showed stable and significant low expression in 16HBE-T and other lung cancer cell lines H460,H1299 and A549,with cyclic characteristics.3.The low expression of circ＿0001839 enhanced the proliferation and migration ability of B[a]P-induced lung cancer cells 16HBE-T,while the proliferation and migration ability of 16HBE-T cells were inhibited by overexpression of circ＿0001839.Circ＿0001839 had a functional role in B[a]P-induced lung cancer cells.4.The single nucleotide polymorphism(SNP)variant of the oncogene MYCL at locus rs1038259890,C>T,occurred in B[a]P-induced lung cancer cells 16HBE-T.Increasing the mutation frequency of MYCL rs1038259890,C>T enhanced the proliferation and migration ability of 16HBE-T and further enhanced the proliferation and migration ability of 16HBE-T after combined interference with circ＿0001839expression,indicating that mutations in MYCL rs1038259890,C>T have a functional in the development of lung cancer cells role.5.During the B[a]P-induced malignant transformation of human bronchial epithelial cells,the expression proportion of C bases at this locus of MYCL rs1038259890 gradually decreased,while the expression proportion of T bases gradually increased,with the fastest decreasing and increasing trend in the interval from the 50 th to 60 th generation of B[a]P exposure,and stabilized at the 80 th generation of B[a]P exposure.The MYCL rs1038259890,C>T mutation occurred in cells exposed to B[a]P in the 60 th generation.6.Knockdown of circ＿0001839 promotes the development of MYCL rs1038259890,C>T mutation,which increases the frequency of this mutation.While overexpression of circ＿0001839 significantly inhibits the mutational progression of MYCL rs1038259890,C>T and reduces its mutation frequency.Conclusion1.Circ＿0001839 was found to be downregulated in B[a]P-exposed human bronchial epithelial cells and lung cancer cells,which played a functional role in B[a]P-induced lung carcinogenesis.2.Mutations in MYCL rs1038259890,C>T enhanced the proliferation and migration of B[a]P-exposed lung cancer cells.3.Circ＿0001839 was involved in the regulation of lung carcinogenesis by regulating the mutation frequency of MYCL rs1038259890,C>T and influencing its mutational progression.