A Chimeric Protein PTEN-L-p53 Enters U251 Cells To Repress Proliferation And Invasion

PTEN,a well-known tumor suppressor,dephosphorylates PIP3 and inhibits AKT activity.A translational variant of PTEN has been identified and termed PTEN-Long?PTEN-L?.The additional 173amino acids?PTEN-L leader?at the N-terminal constitute a potential signal peptide.Differing from canonical PTEN,PTEN-L is secreted into the extracellular fluid and re-enters recipient cells,playing the similar roles as PTEN in vivo and in vitro.This character confers the PTEN-L a therapeutic ability via directly protein delivering instead of traditional DNA and RNA vector options.In the present study,we employed PTEN-L leader to assemble a fusion protein,PTEN-L-p53,inosculated with the transcriptional regulator TP53,which is another powerful tumor suppressor.We overexpressed PTEN-L-p53 in HEK293T cells and detected it in both the cytoplasm and nucleus.Subsequently,we found that PTEN-L-p53 was secreted outside of the cells and detected in the culture media by immunoblotting.Furthermore,we demonstrated that PTEN-L-p53 freely entered the cells and suppressed the viability of U251cells(p53^R273H,a cell line with p53 R273H-mutation).PTEN-L-p53 is composed of endogenous protein/peptide bearing low immunogenicity,and only the junction region between PTEN-L leader and p53 can act as a new immune epitope.Accordingly,this fusion protein can potentially be used as a therapeutic option for TP53-abnormality cancers.Purpose1.To identify the overexpression of PTEN-L-p53 gene in HEK293T cells.2.To identify the re-enter ability of PTEN-L-p53 protein3.To identify that PTEN-L-p53 can enter cellular nucleus to play its biological function like p53.4.To identify the R273H mutation of TP53 in U251cell line that harbors histidine instead of arginine.5.To identify that PTEN-L-p53 re-enters U251 cells to repress the proliferation and metastasis.MethodFirstly,the pSecTag2 A-PTEN-L-p53,pcDNA3.1-TP53 expression plasmids were constructed and successfully overexpressed in HEK293T cells,which was testified by the immunoblotting assay.Subsequently,in order to detect its secretion ability,Ni-NAT agarose beads were used to purify PTEN-L-p53 from both the cell lysate and culture media of HEK293T cells.Western blot testified that PTEN-L-p53 re-entered U251 cells.Moreover,subcellular location of PTEN-L-p53 was detected by immunoblotting via Nuclear and Cytoplasmic Protein Extraction.In order to correct the TP53-distortion carcinogenesis,U251 cell line was selected to serve as a research target,where a mutated p53^R273H?mut-p53?have lost its tumor-suppressive functions.TP53 and PTEN-L-p53 gene were transfected into U251 cells.The proliferation,migration and invasion ability of the transfected cells were detected by cell counting,CCK8,wound-healing and transwell assay.Lastly,PTEN-L-p53 was added to the culture media of U251 cells,and the cell vitality were detected via the described mehods.ResultsIn the present study,the pSecTag2A-PTEN-L-p53 and pcDNA3.1-TP53 plasmids were successfully overexpressed in HEK293T cells.PTEN-L-p53 was purified from both the cell lysate and culture media of HEK293T cells via Ni-NAT agarose beads.Results indicated that PTEN-L-p53 was exported from the donors.Subsequently,PTEN-L-p53 was added into the culture medium of U251 cells.It is clearly that PTEN-L-p53 can re-enetr the cells.After HEK293T cells were treated with PTEN-L-p53,the cell lysates were isolated via Nuclear and Cytoplasmic Protein Extraction.Results demonstrated that PTEN-L leader did not have an adverse effect on the nuclear p53 function.DNA reverse sequencing results validated that U251 cell line expressed a mutated TP53^R273H.R273H mutation is that harbors histidine instead of arginine.U251 cell line was selected to serve as a research target,the cell proliferation and metastasis was significantly suppressed by TP53 and PTEN-L-p53 gene transfection.Lastly,PTEN-L-p53 was added into U251 cells and significantly repressed the cell vitality.Conclusions1.PTEN-L leader drives p53 re-entry to U251 cells.2.PTEN-L-p53 permeates the membrane barriers to locate in the both cellular cytoplasm and nucleus.3.PTEN-L-p53 represses the proliferation and metastasis of U251 cells.