Effects Of Benzo(a) Pyrene On The Expression Of Reporter Genes Driven By The Promoter Region Of Twist Gene

ObjectiveThe Twist gene promoter driving luciferase reporter gene expression plasmid will be constructed,the successfully constructed plasmid will be transiently transfected into non-small cell lung cancer cell line A549 and human embryonic kidney cell line HEK293,and then the transfected cells will be treated with different concentrations of benzo(a)pyrene.This study aims to explore the effects of benzo(a)pyrene exposure on the expression of reporter gene driven by the Twist gene promoter region,which may provide an experimental evidence for understanding the molecular mechanism of benzo(a)pyrene exposure inducing Twist gene expression to promote lung cancer metastasis.Methods1.Using molecular cloning technology to design primers with KpnI and XhoI cleavage sites based on the sequence of the Twist(human)gene promoter region newly published on the NCBI(https://www.ncbi.nim.nih.gov/)website,amplifying the Twist gene promoter sequence of 451 bp in length by PCR technology,and connecting luciferase reporter vector pGL3-basic with KpnI and XhoI restriction sites and amplified fragment.The recombinant plasmid was named as pGL3-TWIST-promoter-luc.The methods including bacterial PCR,double digestion and sequencing of the recombinant plasmid were used to check whether the pGL3-TWIST-promoter-luc recombinant plasmid was successfully constructed.2.Using transient transfection technology,the successfully constructed pGL3-TWIST-promoter-luc recombinant plasmid and internal reference plasmid were transiently transfected into non-small cell lung cancer cell line A549 and human embryonic kidney cell line HEK293.3.After the cells were transfected for 24 h,the cells were treated with different concentrations of benzo(a)pyrene(0 μM、2 μM and 4 μM),The solvent control was dimethyl sulfoxide(DMSO,0.1%V/V).Three parallel wells were set up for each group.The luciferase activity was detected by the dual luciferase reporter gene experiment.Results1.Using HEK293 cell genomic DNA as a template,the length of the Twist gene promoter region was successfully amplified by PCR reaction with a length of 451 bp.2.The Twist gene promoter driving dual luciferase pGL3-TWIST-promoter-luc recombinant plasmid was successfully constructed,which was confirmed by bacterial solution PCR,double enzyme digestion and sequencing identification of the recombinant plasmid.3.Dual luciferase experiment results showed that the luciferase activity was significantly increased in benzo(a)pyrene exposure group as compared with negative control group in non-small cell lung cancer cell line A549 and human embryonic kidney cell line HEK293(P<0.05).Moreover,the activity of luciferase was increased with the increased concentrations of benzo(a)pyrene.ConclusionThe pGL3-TWIST-promoter-luc recombinant plasmid was successfully constructed and benzo(a)pyrene exposure induced the expression of reporter gene driven by Twist gene promoter in A549 cells and HEK293 cells.