Discovery And Characterization Of A Novel Splice Variant Of The P53 Gene In Jurkat Cells

Objective: p53(35)E4p gene is a novel splice variant of the p53 tumor suppressor gene obtained from Jurkat cells.In this study,we analyzed its sequence and explored whether it expressed protein products;HEK-293 T cells transfection experiment was performed by constructing a recombinant eukaryotic expression vector pIRES2-p53(35)E4p to observe whether forced expression of the p53(35)E4p gene would affect HEK-293 T cells.It will lay the foundation for further research on p53(35)E4p gene and and provide additional insight into the biological function of the TP53 gene.It is hoped to provide some guidance for the diagnosis,prognosis and treatment of tumor.Methods:1.In order to investigate the mutation of p53 gene in Jurkat cells,the CDS region of p53 gene in Jurkat cells was specifically amplified,and the amplified products were sequenced by Sanger,Sequence analysis showed the existence of novel splice variant p53(35)E4p.We performed a sequence analysis of it and Western Blot was performed to detect the expression of p53(35)E4p in Jurkat cells;2.The recombinant eukaryotic expression vector pIRES2-p53(35)E4p wastransfected into HEK-293 T cells by calcium phosphate cell transfection kit.And the expression of p53(35)E4p gene or p53 protein in HEK-293 T cells was detected by Western Blot and immunofluorescence assay;3.After the plasmid pIRES2-p53(35)E4p was introduced into HEK-293 T cells,MTT,PI staining and cell cycle experiments were used to detect the effect of the forced expression of p53(35)E4p on the proliferation,mortality and cell cycle of HEK-293 T cells;4.After p53(35)E4p was transfected into HEK-293 T by calcium phosphate or lipofectamine transfection,we observed the difference of EGFP(Enhanced Green Fluorescent Protein)expression between them,and the transfection efficiency,fluorescence intensity and mean fluorescence intensity(MFI)were assessed by flow cytometry;5.The transcriptome analysis of HEK-293 T cells treated by the two transfection methods was performed to explore the reason of the difference of EGFP expression between the two transfection methods.Result:In this study,we identify a novel mRNA variant of the TP53 gene generated by alternative splicing in the Jurkat leukemia cell line.Because this splice variant has a 200 bp sequence excision in exon 4,it was named p53(35)E4p.To further understand the biological function of p53(35)E4p,expression analysis and transfection experiments were performed.No protein product of p53(35)E4p was identified,but it can enhance the expression level ofa 25 k Da protein binding to anti-p53 antibody;After transfection into HEK-293 T cells expressing wild-type p53 protein,p53(35)E4p exhibited an inhibitory effect on cell proliferation and promoted cell death.Interestingly,p53(35)E4p was found to significantly enhance expression of EGFP downstream of the h-CMV promoter.Transcriptome analysis showed that the genes related to DNA binding and regulation of transcription by RNA polymerase II increased in cells transfected with p53(35)E4p.In addition,there were significant changes in the expression of a large number of unannotated genes,indicating that p53(35)E4p may significantly affect cell metabolism and regulate gene expression.The reason for the different expression of EGFP after lipofectamine transfection with p53(35)E4p,we speculate that it is because lipofectamine induced the occurrence of intracellular NMD,which led to the partial metabolism of p53(35)E4p's mRNA,thereby inhibiting the function of p53(35)E4p gene to promote the expression of exogenous reporter gene EGFP,which resulted in the difference of fluorescence intensity between the lipofectamine transfected group and the calcium phosphate transfected group.Conclusion:1.p53(35)E4p gene is a novel splice variant of TP53,because this variant has a 200 bp sequence excision in exon 4,it was named p53(35)E4p.2.No protein product of p53(35)E4p was identified,but it can enhance the expression level of a protein binding to anti-p53 antibody;3.p53(35)E4p may affect the proliferation of HEK-293 T cells by inducingG1 arrest and cell death.4.Transfection of p53(35)E4p enhances expression of the exogenous EGFP reporter gene.5.Gene exression in HEK-293 T cells is affected by p53(35)E4p,indicating that p53(35)E4p may significantly affect cell metabolism and regulate gene expression.6.Lipofectamine may inhibit the function of p53(35)E4p gene to promote the expression of exogenous reporter gene EGFP by inducing the occurrence of NMD in cells.