Effect Of P53Binding Element On Regulation Of NOD8Gene

Objective:In this study, we observe EGFP drived by the promter of NOD8gene with or without p53binding site in HEK293cell. Then, observe the expression of NOD8mRNA and protein inHEK293cell after transfected EGFP-NOD8fusion protein recombination plasmid with orwithout p53binding site [pNOD8(760bp)-EGFP-NOD8、 mpNOD8(750bp)-EGFP-NOD8];Furthermore, we observe PFT-α (pifithrinalpha, PFT-α) inhibitor of p53whethercan infect EGFP and NOD8protein’s expression. to investigate the role of P53binding elementin regulation of NOD8Methods:It is found that human and chimpanzee both have p53binding site in their core promoterregion of NOD8gene with bioinformation method.In this study, we transfect HEK293cell withpNOD8(760bp)-EGFP and mpNOD8(750bp)-EGFP,after24h we observe EGFP expressionunder the inverted fluorescence microscope.Then,we observe EGFP expression under theinverted fluorescence microscope after that the recombination pNOD8(760bp)-EGFP aretransiently transfected into cell line HEK293. before transfection HEK293cell are treated withdifferent concentration of PFT-α inhibitor of p53for24hours. We also transfect HEK293cellWith pNOD8(760bp)-EGFP-NOD8and mpNOD8(750bp)-EGFP-NOD8By JetPeiT M,theplasmid have or havn’t p53binding site in NOD8gene promoter which can drive EGFP-NOD8fusion protein expression.And HEK293cell are treated with PFT-α inhibitor of p53for24hoursin transfection groups of pNOD8(760bp)-EGFP-NOD8and mpNOD8(750bp)-EGFP-NOD8.Then we use RT-PCR and Western blot to detect the expression of NOD8mRNA andprotein.In addition, we use chromatin immunoprecipitation (ChIP) to observe whether p53canbinding promoter of NOD8gene. Result:The EGFP expression driven by recombination plasmid without p53binding site wasobvious decrease compared with recombination plasmid with p53binding site.In a dose-dependent manner, PFT-α down-regulated the expression of GFP in cells transfectedwith pNOD8(760bp)-EGFP plasmids via inhibiting the function o f P53.The results ofChIP confirmed that P53bound to the promoter of NOD8. The mRNA expression ofNOD8in the cells transfected with pNOD8(760bp)-EGFP-NOD8was stronger than that inthe cells transfected with control vector pEGFP-C2(P<0.05). Furthermore, the mRNAexpression of NOD8was reduced in HEK293cells transfected with the mutated plasmidmpNOD8(750bp)-EGFP-NOD8(before transfected treated with PFT-α)compared with thecells transfected with pNOD8(760bp)-EGFP-NOD8. Meanwhile, PFT-α inhibited themRNA expression of NOD8in the cells transfected with pNOD8(760bp)-EGFP-NOD8ina concentration dependent manner, and PFT-α at concentration of90μmol/L was the mosteffective in inhibiting NOD8mRNA expression (P<0.01). As expected, the proteinexpression of NOD8in pNOD8(760bp)-EGFP-NOD8group significantly increasedcompared with that in pNOD8-C2group, the protein expression of NOD8in mpNOD8(750bp)-EGFP-NOD8group+PFT-α group or pNOD8(760bp)-EGFP-NOD8+PFT-αgroup was dramatically decreased compared with that in pNOD8(760bp)-EGFP-NOD8group.Conclusion:1.The EGFP expression driven by recombination plasmid without p53binding site wasobvious decrease compared with recombination plasmid with p53binding site. The resultsindicate that P53binding element can infect the NOD8promter.’s efficiencies.2.PFT-α can down-regulated the expression of GFP in HEK293cells transfected withpNOD8(760bp)-EGFP plasmids via inhibiting the function of P53. 3The results of ChIP confirmed that P53bound to the promoter of NOD8.4P53binding site in the promoter of NOD8can up-regulation of expression ofNOD8mRNA and NOD8protein.. These results indicate that P53binding element play apositive role in the regulation of NOD8gene.