Expression And Functional Analysis Of TCF4 Isoforms In Human Glioma Cell Lines

Objective:Gliomas are the most common intrinsic brain tumors.They are characterized by high morbidity,high recurrence rates,high mortality and low cure rate.Recently,aberrant regulation of Wnt signaling pathway plays a vital role in the process of glioma and malignant progression.Compared to the normal brain,(3-catenin and TCF4 are up-regulated in gliomas,inhibiting cell proliferation and inducing cell apoptosis.TCF4 is reported to exhibit high expression in renal carcinoma,colorectal cancer,hepatocellular carcinoma and brain tumor.Abnormal rise of TCF4 stimulating downstream target genes is the common early event in tumorigenesis.The human TCF4 gene comprises of 17 exons.Recent studies showed that TCF mRNAs were subject to alternative splicing to form different isoforms,which are also important in regulating transcription in Wnt signaling pathway and associated with tumorigenesis.Different TCF4 isoforms have been found in various types of cancer including renal carcinoma,colorectal cancer and brain tumor.However,the role of TCF4 isoforms in the process of gliomas remains to be elucidated.In our research,wo plan to clone and identify TCF4 isoforms in three human glioma cells,and find the effects of cell proliferation,cell apoptosis and cell migration in U251 cells.Methods:Human glioma cell lines U251,A172 and U-87MG were maintained in DMEM(Gibco)supplemented with 10%fetal bovine serum(FBS,Gibco),100 U/ml of penicillin and 100 μg/ml of streptomycin.TCF4 gene amplification was applied in three types of glioma cell lines by RT-PCR.Sequence alignments were performed with TCF4 genome deposited in Genbank by BLAST.Plasmid construction was done with three isoforms we chosen,and was transfected to U251 cell lines.After that,we test the proliferation、apoptosis and migration of U251 cells by MTT assay、flow cytometry and wound healing assay.The statistical analysis was done at last.Results:In our study,13 different TCF isoforms were expressed in U251 cells,among which 3 isoforms(vx35,vx18 and v7)were in the front rank after blasting with TCF genome the NCBI database.19 different TCF isoforms were expressed in A172 cells,among which 3 isoforms(vx18,vx27 and vx21)gain top scores.12 different TCF isoforms were expressed in U-87MG cells,among which 3 isoforms(vx27,vx18 and vx21)were on the top.In MTT assay,U251 cells transfected with pVITRO-neo-TCF4vx35,pVITRO-neo-TCF4vxl8 and pVITRO-neo-TCF4vx7 decreased cell proliferation compared to empty vector control at 48 h and 60 h(P<0.001).In flow cytometry,U251 cells transfected with pVITRO-neo-TCF4vx35,pVITRO-neo-TCF4vxl8 and pVITRO-neo-TCF4vx7 induced 31.59%,19.60%and 38.43%more apoptosis than empty vector control(6.67%)(P<0.001).In wound healing assay,U251 cells over-expressed pVITRO-neo-TCF4vx35,pVITRO-neo-TCF4vxl8 and pVITRO-neo-TCF4vx7 resulted in increased cell migration but the migration was lower than control.Conclusion:In conclusion,the TCF4 isoforms in three human glioma cells lines were cloned and identified in the present study.The functions and properties of TCF4 isoforms were analyzed in U251 cells.The results showed TCF4 isoforms inhibit cell proliferation and induce the cell apoptosis and migration.We propose that this mechanism of Wnt/β-catenin-mediated TCF isoforms transcription regulates target genes contribute to tumorigenesis of glioma.