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AIM:To evaluate the inhibition effect of HCV NS5A on p53transactivation on p21 promoter and explore its possiblemechanism for influencing p53 function.METHODS:p53 function of transactivation on p21 promoterwas studied with a luciferase reporter system in which theluciferase gene is driven by p21 promoter,and the p53-DNAbinding ability was observed with the use of electrophoreticmobility-shift assay(EMSA).Lipofectin mediated p53 orHCV NS5A expression vectors were used to transfecthepatoma cell lines to observe whether HCV NS5A couldabrogate the binding ability of p53 to its specific DNAsequence and p53 transactivation on p21 promoter.Western blot experiment was used for detection of HCVNS5A and p53 proteins expression.RESULTS:Relative luciferase activity driven by p21 promoterincreased significantly in the presence of endogenousp53 protein.Compared to the control group,exogenousp53 protein also stimulated p21 promoter driven luciferasegene expression in a dose-dependent way.HCV NS5Aprotein gradually inhibited both endogenous andexogenous p53 transactivation on p21 promoter withincrease of the dose of HCV NS5A expression plasmid.Bythe experiment of EMSA,we could find p53 binding to itsspecific DNA sequence and,when co-transfected withincreased dose of HCV NS5A expression vector,the p53binding affinity to its DNA gradually decreased and finallydisappeared.Between the Huh 7 cells transfected withp53 expression vector alone or co-transfected with HCVNS5A expression vector,there was no difference in thep53 protein expression.CONCLUSION:HCV NS5A inhibits p53 transactivation onp21 promoter through abrogating p53 binding affinity toits specific DNA sequence,it does not affect p53 proteinexpression.
AIM: To evaluate the inhibition effect of HCV NS5A on p53 transactivation on p21 promoter and explore its possible mechanism for influencing p53 function. METHODS: p53 function of transactivation on p21 promoter was studied with a luciferase reporter system in which the luciferase gene is driven by p21 promoter, and the p53-DNA binding was observed with the use of electrophoretic mobility-shift assay (EMSA). Lipofectin mediated p53 or HCV NS5A expression vectors were used to transfect hepatoma cells lines to observe whether HCV NS5A couldabrogate the binding ability of p53 to its specific DNA sequence and p53 transactivation on p21 promoter. Western blot experiment was used for detection of HCV NS5A and p53 protein expression .RESULTS: Relative luciferase activity driven by p21 promoterincreased significantly in the presence of endogenousp53 protein .Compared to the control group, exogenousp53 protein also stimulated p21 promoter driven luciferasegene expression in a dose-dependent way. HCV NS5Aprot ein solid inhibited both endogenous an trans spontaneous p53 transactivation on p21 promoter withincrease of the dose of HCV NS5A expression plasmid. the experiment of EMSA, we could find p53 binding to it specific DNA sequence and, when co-transfected within HCV based NS5A expression vector, the p53binding affinity to its DNA dest decreased and finally dosedappeared.Between the Huh 7 cells transfected withp53 expression vector alone or co-transfected with HCVNS5A expression vector, there was no difference in the p53 protein expression.CONCLUSION: HCV NS5A inhibits p53 transactivation onp21 promoter through abrogating p53 binding affinity toits specific DNA sequence, it does not affect p53 proteinexpression.