Regulation Of Pin1Expression By Dexamethasone

Objective Huh-7cells were treated with dexamethasone,and subjected to measurement of Pinl mRNA and protein expression levels at different time points after treatment. We cloned the promoter of Pin1gene by PCR, constructed its luciferase report plasmid pPinl-Luc, and evaluated the effect of dexamethasone on the transcriptional activity of Pinl promoter.Methods1、 Huh-7cells were treated with dexamethasone, and their mRNA and protein levels of Pinlwere measured by quantitative real-time PCR and Western-blot analysis, respectively, at0h、3h、6h、12h、24h after treatment;2、Genomic DNA was extracted from WT mouse, and the Pinl gene promoter was amplified by PCR, and then cloned into vector pGL4.0to obtain the recombinant plasmid pPinl-Luc.which was identified by restriction enzyme digestion and DNA sequencing.3、Huh-7cells were transfected with pPin1-Luc or control plasmid pGL4.0by Effectene,and treated with or without DEX.72h after transfection,Huh-7cells were lysed and harvested to test the luciferase activity using Dual-Luciferase Reporter Assay System.Results1、After treatment of Huh-7cells with dexamethasone, Pinl mRNA levels reached a peak at3h (P<0.01), and its protein expression levels were highest at6h (P<0.01);2、Restriction enzyme digestion and DNA sequencing result confirmed the recombinant plasmid, pPinl-Luc were correctly constructed;3、The recombinant plasmid pPinl-Luc and pGL4.0were successfully transfected into Huh-7cells; Compared with control,the activities of luciferase reporter genes significantly increased when stimulated with dexamethasone72h after Huh-7cells were transfected with pPinl-Luc(P<0.05)Conclusion Dexamethasone can enhance the Pinl mRNA and protein levels in liver cancer cells Huh-7. The pPin1-Luc was successfully constructed. Dexamethasone can enhance the transcriptional activity of Pinl gene promoter.