The Preliminary Study On The Regulation Mechanism Of Mouse GSTK1Gene Promoter

Part1. Construction and functional identification of mouse GSTK1promoter luciferase reporter gene plasmidsObjective：To construct luciferase reporter gene plasmids driven by mouse GlutathioneS-transferase kappa (mGSTK1) promoter and analyze their activities.Methods：The mouse GSTK1gene promoter fragment was amplified from mouse genomicDNA by PCR. Product of PCR was inserted into the pGL3-Basic luciferase vector, andgenerated a luciferase reporter gene plasmid, pGL3-mGSTK1-promoter-2046. Threereporter gene constructs containing various segments of the mGSTK1promoterregions,pGL3-mGSTK1-promoter-936.pGL3-mGSTK1-promoter-228and.pGL3-mGSTK1-promoter-76were generated by PCR using pGL3-mGSTK1-promoter-2046as a template.The constructed reporter plasmids containing different length of mGSTK1promoter weretransiently transfected into3T3-L1and HEK293, and the luciferase activities weredetected48h after transfection. Results：All constructed plasmids were confirmed byrestriction enzyme digestion and sequence analysis. Transient transfection assay in twocells approved that pGL3-mGSTK1-promoter-2046、 pGL3-mGSTK1-promoter-936andpGL3-mGSTK1-promoter-228showed high transcriptional activity and pGL3-mGSTK1-promoter-76showed the lowest activity.Conclusion：The luciferase reporter gene plasmids driven by mouse GSTK1promoter weresuccessfully constructed, and-263to-111region relative to the translation start site isimportant for the mGSTK1gene promoter. Part2. Effect of rosiglitazone on mRNA level of GSTK1in vitroObejective:To observe the effect of rosiglitazone on GSTK1transcriptional level in3T3-L1adipocyte and mice fat tissue, and analyze the effect of peroxisome proliferator-activedreceptor gamma (PPARγ) on the luciferase activity of pGL3-mGSTK1-promoter-2046.Methods:Differentiated3T3-L1adipocytes and mice fat tissue were treated with the vehicle control(DMSO) or rosiglitazone for24hours and samples were collected after24h treatment. RelativemRNA levels of GSTK1and C/EBPαwere determined by real-time PCR. The reporter geneconstruct pGL3-mGSTK1-promoter-2046was cotransfected with expression plasmidspCMV-PPARγ and pCMV-RXR into HEK293cells.48hours after transfection, luciferasereporter assays were performed using Double Luciferase Assay System.Resultes:In3T3-L1adipocytes and mice fat tissue, GSTK1and C/EBPα gene mRNA werestimulated by rosiglitazone. There was no effect of PPARγ on the luciferase activity of reportergene construct pGL3-mGSTK1-promoter-2046.Conclusion:The GSTK1gene mRNA was upregulated by rosiglitazone in vitro. There might be noPPARγ binding site between-2081and-35upstream of GSTK1gene translation start site and theincreased GSTK1mRNA level might be associated with the elevated expression of C/EBPα.