Transcriptional Regulation Of P53 In Glioma On The Cancer-testis Antigens OY-TES-1

Objective:Our previous study found that glioma had high expression of cancer-testis antigens OY-TES-1 and was associated with p53 expression.This study aims to understand the effect of p53 on the expression of OY-TES-1 in glioma,and understand the transcriptional regulation of p53 as a transcription factor on the promoter of OY-TES-1,so as to provide experimental basis for the preliminary clarification of the expression mechanism of OY-TES-1 in glioma.Methods:（1）To construct and verify glioma cell models with different p53 states:firstly,glioma cell lines（U87MG,A172）of wild-type p53(p53^Wt)were selected,and the p53^Wt gene was knocked out by CRISPR-Cas9 gene editing technology to construct a cell model with p53 gene deletion(U87MG-p53^ko,A172-p53^ko).Then,lentivirus vectors(Lv-p53^Mt175and Lv-p53^Mt248)of two common p53mutation sites(p53^Mt175and p53^Mt248)were constructed and transferred to the p53^ko glioma cell lines,respectively,to construct the glioma cell model with p53mutation(U87MG-p53^Mt175and U87MG-p53^Mt248).(A172-p53^Mt175and A172-p53^Mt248).q RT-PCR and Western Blot were used to detect p53 from m RNA and protein levels,as well as the protein levels of p21,the downstream target gene of p53,and to evaluate whether the glioma cell model with p53deletion/mutation was successfully constructed.（2）The expression of OY-TES-1 in cells with different p53 states was detected:the expression of OY-TES-1 in p53^Wt glioma cell line,p53^ko glioma cell line and p53^Mtglioma cell line was detected from RNA and protein levels by q RT-PCR and Western Blot methods,respectively.（3）Prediction of transcription factor binding sites on OY-TES-1 promoter:two online transcription factor prediction tools（Mat Inspector,PROMO）were used to predict the core promoter region of OY-TES-1（-184 to+66NT）confirmed by the previous research group,so as to search for potential transcription factor binding sites.（4）Design and construction of OY-TES-1 core promoter reporter vector:OY-TES-1 core promoter sequence（-184 to+66NT）and the p53 binding site mutation OY-TES-1 core promoter sequence were inserted into the pGL3-basic vector at the Kpn I and Hind III restriction sites,respectively,and the following pGL3 recombinant plasmids were constructed:the recombination plasmid of OY-TES-1 core promoter（pGL3-OY）,the recombination plasmid of the OY-TES-1 core promoter（pGL3-OY-M1）,the recombination plasmid of the OY-TES-1 core promoter（pGL3-OY-M2）,and the recombination plasmid of the p53 binding site（pGL3-OY-M3）.（5）Luciferase assay:the constructed pGL3 recombinant plasmid was transfected with Lipofectamine（?）2000 reagent.The experiment was divided into two parts:first,pGL3-OY was transferred into p53^Wt cells,p53^ko cells and p53^Mtcells,respectively,to detect luciferase activity and understand the influence of p53 in different states on the activity of OY-TES-1 promoter.Second,pGL3-OY,pGL3-OY-M1,pGL3-OY-M2 and pGL3-OY-M3 were transfected into p53^Wtcells,respectively,to detect luciferase activity and to find out whether the activity of the OY-TES-1 promoter was related to the mutation of the p53binding site.（5）Ch IP-q PCR experiment p53 with the OY-TES-1 promoter:extract nuclear extracts of different p53 status of glioma cell line,will be nuclear extracts with p53 antibody incubation,enrichment and p53 protein in combination with DNA fragments,purification of sediment,through q-PCR amplification OY-TES-1 promoter fragment,validation of p53 with the OY-TES-1 promoter.Results:（1）Successfully constructed p53 deletion/mutant glioma cell lines:p53^kocells were detected by q RT-PCR and Western Blot,and the results showed that p53 m RNA was hardly detectable or negative,p53 protein was negative,and p21 protein level was significantly down-regulated,indicating that p53 gene had been knocked out.The results of p53^Mt cells showed that both p53 m RNA and protein were positive,indicating that the p53 mutant glioma cell line had been successfully constructed.（2）p53 affects the expression of OY-TES-1:q RT-PCR results showed that OY-TES-1 m RNA was down-regulated in p53^Wt cells,while OY-TES-1 m RNA was up-regulated in p53^ko and p53^Mt cells.The expression level of OY-TES-1m RNA in p53^Wt cells was statistically significant compared with OY-TES-1m RNA in p53^ko and p53^Mt cells.Western Blot results showed that,compared with p53^ko and p53^Mt glioma cells,the OY-TES-1 protein in p53^Wt cells was significantly reduced,and the difference was statistically significant.These results suggested that wild-type p53 inhibited the expression of OY-TES-1m RNA and protein,while p53 deletion and mutation enhanced the expression of OY-TES-1 m RNA and protein.（3）OY-TES-1 core promoter region contains two p53 binding site:online transcription factor is applied to forecast the software at the heart of the OY-TES-1 promoter region were analyzed,and the results show that in the area has two p53 binding sites,one of the p53 binding sites with overlapping Sp1binding sites,this suggests the p53 may as a transcription factor transcription regulation OY-TES-1.（4）Successful construction of OY-TES-1 core promoter reporter vector:the constructed pGL3-recombinant plasmids（pGL3-OY,pGL3-OY-M1,pGL3-OY-M2 and pGL3-OY-M3）were used for DNA sequencing,and the sequencing results were blast compared,which showed a complete match with the sequences in Genbank,confirming the successful construction of the vector.（5）p53 affects the activity of OY-TES-1 promoter:first,the luciferase activity of pGL3-OY cells in different p53 states(p53^Wt,p53^ko and p53^Mt)was measured.The results showed that compared with pGL3-Basic,the luciferase activity of p53^Wt cells was decreased,suggesting that the activity of OY-TES-1promoter could be inhibited by wild-type p53.Secondly,pGL3-OY,pGL3-OY-M1,pGL3-OY-M2 and pGL3-OY-M3 were transfected into p53^Wtcells,and the luciferase activity test results showed that compared with the pGL3-OY group,the luciferase activities of pGL3-OY-M1,pGL3-OY-M2 and pGL3-OY-M3 were all significantly increased except for the decreased luciferase activity of U87MG cells after transfection with pGL3-OY-M1.This result indicated that in most cases,the p53 binding site mutation on the core promoter of OY-TES-1 would increase the promoter activity.（6）OY-TES-1 may be the target gene of p53:Ch IP-q PCR showed that p53^Wtcould bind to OY-TES-1 promoter.For mutated p53,p53^Mt175 cannot bind to OY-TES-1 promoter.p53^Mt248 could not bind to OY-TES-1 promoter in different cells.In U87MG cell lines,p53^Mt248 could not bind to OY-TES-1promoter.However,in A172 cell line,p53^Mt248 could bind to OY-TES-1promoter,and the enrichment of OY-TES-1 promoter was different with p53 in different states.Conclusion:p53 as a transcription factor is involved in the transcriptional activation of OY-TES-1 in glioma.