Study On The Modulation Of P53by GPX2and The Mechanism

GPX2is a member of GPX family, which is the mainly enzyme of antioxidant.Previous studies revealed that GPX2play an role in cell proliferation, invasion andmigration. Some studies reported GPX2had anti-apoptotic and anti-inflammatoryfunction. p53was a transcription factor, played an important regulatory role inmaintaining genomic stability and in cell growth, differentiation, proliferation andapoptosis. p53has both pro-and anti-oxidant activities, each of which contributes totumor suppression. We found GPX2may exist the interaction with p53usinghigh-throughput yeast two hybrid, but the molecular mechanism and biologicalsignificance of the interaction had no report. GPX2and p53closely linked to tumordevelopment and oxidative stress in vivo. GPX2may regulate tumor cell behaviorthrough protein-protein interaction with p53, and that provides ideas for pathogenesisand therapy of tumor.Firstly we use co-IP and GST-pull down to confirm the interaction betweenGPX2and p53and analysis of binding domain of GPX2with p53by Co-IP. Then wefound GPX2has an inhibitory effect on the transcriptional activity of p53and itsdownstream target genes by luciferase reporter assay. We also find GPX2candown-regulate the expression of p53and its downstream target genes by real timequantitative PCR and Western Blot. GPX2promote the degradation of p53byhalf-life experiment, however, GPX2has no significant effect on p53ubiquitylation.The effects on cycle were analyzed by FACS, we found knock down GPX2inHCT116p53+/+cells has certain influence to the cycle, cells number in G0/G1phasewas reduced by20%than the control, while knock down GPX2in HCT116p53-/-cells had no effect on cycle, so GPX2affectted cell cycle on a p53-dependent way.For further study the biological function of GPX2, we construct a lentiviral vectorpCDH-GPX2for GPX2expression and another pSicoR-GPX2for GPX2RNAinterference, and gain the lentiviral particles which can stably up/down-regulate theexpression of GPX2. GPX2knockdown likely lead to cell apoptosis. The reason isthat pro-apoptotic protein Bax was activated and anti-apoptotic protein Bcl-2wasinhibited.In conclusion, we discovered that GPX2as a new kind of p53negative regulatory factor for the first time, promoted the degradation of p53protein, inhibitedthe transcriptional activity of p53, down-regulated of p53and its downstream targetgenes’ expression，and impacted on the cycle of tumor through the interactionbetween GPX2and p53. Besides, lentivirus of GPX2was successfully constructed,which establish a basis for further study.