The Preparation Of A New Type Of Eukaryotic Recombinant Protein And Its Chemotherapy Sensitization Effect On Human Glioma Stem Cells

[Object]Glioma is the most common primary tumor of the human brain.It has extremely strong proliferation and migration.The resection of glioma in combination with chemoradiation has demonstrated advantages over best supportive care in prolonging survival.But it still has a high recurrence rate.In recent years,cancer stem cell(TSC)hypothesis has been becoming particularly hot.It proposes that tumor recurrence and metastasis after treated with drugs are closely related to the survival of tumor stem cell.Therefore,the study of TSC has very important significance to the development of more effective anti-tumor drugs.Recent studies have shown that the inappropriate activation of Wnt/?-catenin pathway is involved in tumorigenesis,invasion and migration,which is closely related to tumor stem cells.The survival and maintenance of tumor stem cells depends on the abnormal high expression of the transcription factor complex of ?-catenin/TCF4 in the nucleus.The ?-catenin in the nucleus has to combine with the transcription factor TCF4.Then it can act as the transcription of the target gene.Thus,the destruction of the binding of the transcriptional complex can be used as an effective way to intervene in the formation of tumor stem cells.At present,it is known that the N-terminal 80 aminoacids of the TCF4 are the key binding domains with ?-catenin.In this study,we constructed the protein polypeptide(named NLS-N80-TAT)with a 20 molecular weight by extracting the first 80 amino acids.It was confirmed that the recombinant protein could inhibit the binding of TCF4 to ?-catenin as a competitive inhibitor and the transcriptional activity of TCF /LEF,which also inhibited the proliferation of glioma stem cells and promoted their differentiation,and thus promoted the sensitivity of glioma stem cells to chemotherapy drugs.[Methods]1.Preparation and identification of eukaryotic recombinant protein NLS-N80-TAT(1)The cloning of NLS-His-FLAG-N80-TAT geneThe N80 sequence containing the first 80 amino acids of TCF4 was amplified by high-fidelity PCR amplification kit with pc DNA / Myc-TCF4 vector a template.His-Flag sequence and TAT sequence were introduced into the upstream and downstream primers respectively.NLS sequence was introduced at the N-terminal to obtain a primary PCR product and the final NLS-His-FLAG-N80-TAT sequence was amplified by using the primary PCR product as a template.(2)The transfection and expression of HEK293 cells.pc DNA3.1-NLS-His-FLAG-N80-TAT were transfected into HEK293 cells with liposomal transfection reagent for 48 hours.The recombinant plasmids were treated with neomycin to construct HEK293 cells with the expression of NLS-His-FLAG-N80-TAT genes which were detected by Western blot in HEK293 cells.(3)Separation and Purification of recombinant ProteinNLS-His-FLAG-N80-TAT.The new eukaryotic recombinant protein was identified by His-monoclonal antibody after SDS-PAGE electrophoresis and transferring membrane.2?The double Immunofluorescence staining assay revealed that NLS-N80-TAT could enter the nucleus by and confirmed that NLS-N80-TAT can bind to ?-catenin by Immunoprecipitation technique.3?the Immunoprecipitation technique confirmed that the NLS-N80-TAT could inhibit the binding of endogenous and exogenous ?-catenin / TCF4 transcription complexes.The luciferase reporter gene assay was used to detect the effect of NLS-N80-TAT on the transcriptional activity of TCF / LEF Pathway.4?The effect of NLS-N80-TAT on the expression level of CD133 in glioma stem cells was detected by RT-PCR.Western blotting was used to detect the expression of proliferation-associated protein(c-Myc)and differentiation-related protein(?-?tublin)in glioma stem cells.And the effect of NLS-N80-TAT on the expression level of ?-? tublin in the glioma stem cells by double immunofluorescence staining[Results]1.Eukaryotic recombinant protein NLS-N80-TAT can enter into the nucleus and bind to ?-catenin2.Eukaryotic recombinant protein NLS-N80-TAT can be used as a competitive inhibitor to block the combination between ?-catenin and the N-terminal of TCF4 in order to inhibit the transcriptional activity of TCF / LEF pathway3.Eukaryotic recombinant protein NLS-N80-TAT can inhibit the formation of glioma stem cells and promote their differentiation.4.Eukaryotic recombinant protein NLS-N80-TAT can promote the sensitivity of glioma stem cells to chemotherapy.[Conclusions]The recombinant protein NLS-N80-TAT can be used as a competitive inhibitor to block the binding of TCF4 to ?-catenin,and inhibit the transcriptional activity of TCF / LEF pathway,which also promotes the sensitivity of glioma stem cells to chemotherapeutic drugs.Clinical treatment of glioma has a very broad prospects.