Functional And Mechanistical Characterization Of CPEB2-p53 Negative Feedback Loop In Renal Cancer

As one of the most important tumor suppressor genes,p53 is involved in the regulation of a variety of cytological events in the process of tumorigenesis and development,and has always been the hot spot in oncology research field.As the"guardian of genome",p53 plays a biological role mainly by activating or inhibiting the expression of a series of downstream target genes through its transcription factor activity.Therefore,screening new p53 target genes and studying their roles are of great significance for comprehensively understanding the functional system of p53.In addition,about 50%of tumors have mutations in p53,and in other tumors without p53 mutation,the p53 signaling pathway is often inhibited or inactivated.Therefore,a better elucidation of the upstream molecular mechanisms of regulation of p53 can provide new ideas for understanding the pathogenesis and prevention of cancer.We found thatRNA-binding protein CPEB2 expression positively correlated to p53 activity through microarray analysis.CPEB2 is a member of cytoplasmic polyadenylation element-binding（CPEB）family protein,which can regulate mRNA stability and protein translation by binding with the cytoplasmic polyadenylate elements（CPE）in the 3’-untranslated region（3’UTR）region of target mRNA to control the elongation of the poly（A）tail.It has been reported that there are classical CPE elements in the 3’UTR region of p53 mRNA.Whether p53 directly regulates CPEB2,and whether CPEB2 can bind with the CPE elements to regulate p53 remain to be investigated.In addition,the role and molecular mechanism of CPEB2 in tumors have not been studied systematically,and the role of CPEB2 in the development and progression of renal cancer remains unclear.In this paper,firstly,three different mouse embryonic fibroblast（MEF）cell models(Mdm2^+/+、Mdm2^-/-and Mdm2^m/m)with p53^ERgene knockin background were used to establish the p53 activation induction model.Using this model,new p53target genes were screened by microarray analysis in the absence of exogenous injurious stimulation.A new potential target gene of p53,CPEB2,was screened out.It was confirmed by q RT-PCR and westernblot experiment that the expression of CPEB2 in three kinds of MEF cells showed a trend of low,medium and high,which was correlated with the transcriptional activity of p53.Chromatin immunoprecipitation results showed that p53 can directly bind to CPEB2 promoter.The luciferase assay showed that exogenous p53 enhanced the activity of CPEB2promoter,and the p53 binding site was confirmed by mutation.Further studies showed that CPEB2 expression was upregulated by various stress signals that activated p53.Taken together,these results indicate that CPEB2 is a direct target gene of p53,and p53 upregulates the expression of CPEB2 under normal and stress-induced conditions.Secondly,this study further explores whether CPEB2 can feedback to regulate p53 and related molecular mechanism.Firstly,westernblot and q RT-PCR experiments confirmed that CPEB2 could negatively feedback to regulate the protein and mRNA levels of p53.Immunofluorescence and nuclear-cytoplasmic separation experiments confirmed that CPEB2 mainly localized in the cytoplasm,suggesting that CPEB2 mainly affected the stability of target mRNA and protein translation.It was confirmed that CPEB2 could promote the degradation of p53mRNA by detecting the half-life of p53 mRNA.In addition,cycloheximide（CHX）removal assay showed that CPEB2 inhibited p53 translation,but did not affect the stability of p53 protein.Finally,RNA Immunoprecipitation（RIP）andRNA pulldown experiments confirmed that CPEB2 mainly binds to the two adjacent CPE elements at the end of the 3’UTR region of p53 mRNA through its own RRMs and ZF domain to regulate p53.Thirdly,the role of CPEB2 in the development and progression of renal cancer was studied using renal cancer as a model.Combined with the clinical tissue samples of renal cancer and the relevant data of CPEB2 in the TCGA database,the analysis showed that the expression of CPEB2 in renal cancer tissues was significantly higher than that in adjacent normal tissues.To clarify the biological function of CPEB2,renal cancer cell lines with CPEB2 stable knockdown and overexpression were constructed using lentiviral packaging system.In vitro and in vivo experiments confirmed that CPEB2 promoted the proliferation of renal cancer cells.Further analysis by flow cytometry showed that CPEB2 induced G1/S phase transition and significantly inhibited cisplatin induced cell death.In addition,CPEB2 significantly promoted the migration and invasion of renal cancer cells.Finally,the oncogenic effect of CPEB2 in renal cancer is partially dependent on the negative feedback regulation of p53 function.In summary,this thesis confirmed that CPEB2 is a direct target gene of p53.By contrast,as anRNA binding protein,CPEB2 binds to CPE elements in the 3’UTR region of p53 mRNA and feedback to regulate p53,promoting the degradation of p53 mRNA,forming the negative feedback loop of CPEB2-p53.CPEB2 is highly expressed in renal cancer tissue,which promotes the proliferation,migration and invasion of renal cancer cells,and the oncogenic effect of CPEB2 in renal cancer is partially dependent on the negative feedback regulation of p53 function.This study highlights a novel mechanism by which p53 activity is controled in tumor cells and offers new therapeutical potential to treat renal cancer.