The Role Of P53 Target Gene Rap2a In Mechanism Of Human Tumor Migration And Invasion

Context:The transcription factor p53 is well known as a potent suppressor of tumor development and plays a central role in tumor prevention.Upon various stresses,such as DNA damage and abnormal growth signals,p53 binds to chromatin and activates or represses numerous downstream genes that elicit many cellular outcomes,such as cell cycle arrest,apoptosis differentiation and DNA repair.Recent evidences suggest that p53 may contribute to the regulation of cell invasion and migration.Rap2a,a member of the small GTPase superfamily,mediate diverse cellular events such as cell proliferation,adhesion and migration through various signaling pathways.It has been reported that Rap proteins affect the tissue invasiveness and metastasis of various human cancers.Objective:To investigate the relationship between p53 and Rap2a and the effect of Rap2a on migration and invasion of tumor cells.Methods:U20S and H1299 cells were treated with 5 nM actinomycin D(ActD)for 24 h,and the gene expression of Rap2a was measured by RT-PCR analysis.Cells were treated with different dose of ActD(0,1,5,10 nM),the protein expression of Rap2a,P53 and p21 were observed.Cells were treated with 5 nM ActD at different times(Oh,4h,8h,12h,16h,20h,24h),the protein expression of Rap2a,P53 and p21 were observed.Rap2a and P53 protein levels were detected after transiently transfection of the chemically-synthetic siRNA targeting p53.We used chromatin immunoprecipitation(ChIP)to investigate whether p53 regulate directly Rap2a at transcriptional level.The protein expression of Rap2a was detected by immunoblot analysis in human tumor cell lines.The viability and apoptosis of the cells were measured by CCK-8 assay and Annexin V-FITC binding assay.Meanwhile,the protein levels of Bcl-2,Bax and pro-caspase3 were measured by western blot.Furthermore,the abilities of migration and invasion were evaluated by wound healing assay and Transwell test.We also detected MMP2 and MMP9 enzyme activity by gelatin zymography.And the expression of FAK,P-catenin,Akt,p-Akt was detected by immunoblot analysis and the mechanisms of Rap2a on migration and invasion was further investigated.Results:mRNA level of Rap2a increased significantly in U20S cell lines when compared with control group.In contrast,there was no significant difference in H1299 cells.The protein levels of Rap2a,P53 and p21 increased in U20S cells treated with different concentrations of actinomycin D(0,1,5,10 nM)for 24h.In addition,similar induction of Rap2a was observed in the cells treated with 5 nM actinomycin D at different time point.However,increased Rap2a expression was not observed in H1299 cells following actinomycin D treatment,which is consistent with the result of RT-PCR analysis.The induction of Rap2a by p53 was inhibited by p53 siRNA.Upon DNA damage,p53 directly binds to the promoter of Rap2a and activates its transcription.Further studies revealed that Rap2a is up-regulated in many types of tumors.In addition,the wound healing assay and Transwell assay indicated that the ectopic expression of Rap2a promotes cancer cell invasion and migration,whereas knockdown of Rap2a inhibits cell invasion and migration.Correspondly,enzyme activity of MMP2 and MMP9 also changed.MTT assay and Annexin V/PI assay showed that Rap2a has no effect on the proliferation of cancer cells.In addition,no significant change in the expression levels of Bcl-2,Bax and caspase-3 were found in Rap2a-treated cancer cells.Activated Rap2a promotes the phosphorylation level of AKT.In contrast,knockdown of Rap2a decreased the phosphorylation level of AKT.Activation of Rap2a has no effect on the protein expression of FAK,P-catenin.Conclusion:Our results suggest that Rap2a is a novel target of p53 and is induced upon DNA damage in a p53-dependent manner.The expression levels of Rap2a were predominantly increased in human tumor cell lines.The ectopic expression of Rap2a promotes cancer cell invasion and migration,whereas knockdown of Rap2a inhibits cell motility.The pro-migration function of Rap2a might be mediated via the regulation of AKT pathway.Thus,it suggests that Rap2a and p53 form a portion of a regulatory transcriptional network that appears to play a critical role in regulating cancer cell invasion and migration.