Endogenous FOXP3 Inhibits Cell Proliferation, Migration And Invasion In Glioma Cells

Background: The transcription factor forkhead box P3(FOXP3) was considered as a specific transcription factor in regulatory T cells(Tregs), and it has been demonstrated to play important roles in the development and function of Tregs. In addition, studies had recently demonstrated that FOXP3 also expressed in some tumor cells and epithelial cells. Our previous studies have found that FOXP3 also expression in glioma cells, however, the exact role and molecular mechanism of FOXP3 function in glioma’s cells are still unclear.Objectives: This study aims to elucidate the influence of FOXP3 on the biological behavior of proliferation, apoptosis, migration and invasion in human glioma cell lines U87 and LN229. To clarify the functional of FOXP3 in glioma cells, and further deepen the understanding of the molecular and molecular basis associated with the development of glioma.Methods: 4 plasmids containing different sequences of shRNA targeting FOXP3 gene(sh RNA1~4) and 1 plasmids containing random sequences(Scrambled) and overexpression of FOXP3 plasmid(p CMV6-FOXP3-GFP) and its load control plasmid(p CMV6-Empty-GFP) were transfected into human glioma U87 cells and LN229 cells mediated by Lipofectamine 2000. And transfection efficiency was observed in an inverted fluorescence microscope. The expression of FOXP3 protein was detected by Western blot to screening the most effective sh RNA plasmid. Cell proliferation of U87 cells and LN229 cells were detected by CCK-8 assay. Flow Cytometry(FCM) was adopted to analyze quantitatively the cell cycle and apoptotic cells in each group. Expression of proapoptotic proteins of caspases-3 and caspases-7 was investigated by Western blot. Migration assay was performed to detect cell migration, and cell invasion assay was performed using Transwell chambers. Application of SPSS software analysis experimental data obtained, P < 0.05 as the difference was statistically significant.Results:(1) 72 h after transfection a large number of fluorescent cells are observed by inverted fluorescence microscope in each group, and transfection efficiency is highest.(2) At 72 h after transfection, the expression of FOXP3 protein was most obviously suppressed by sh RNA-1 sequence comparing with other sh RNA sequences. And the overexpression vector having the significant efficiency in up-regulation FOXP3 expression(P <0.05).(3) CCK-8 assay demonstrated that over-expression of FOXP3 in glioma cell U87 and LN229 inhibited cell proliferation, down-regulation of the FOXP3 promoted cell growth(P <0.05).(4) Flow cytometry analysis showed that up-regulation of FOXP3 occurred significant apoptosis, down-regulation of the FOXP3 inhibited cell apoptosis(P <0.05).(5) Up-regulation of FOXP3 increased the protein levels of pro-apoptotic molecules caspases-3 and caspases-7, down-regulation of the FOXP3 reduced its expression(P <0.05).(6) PI staining detected by flow cytometry-cycle analysis revealed that up-regulation of FOXP3 resulted in a significant increase in the G0/G1 fraction and the S fraction was significantly decreased, indicated that cell cycle was arrested(P <0.05).(7) Migration and invasion assay demonstrated that over-expression of FOXP3 reduced cell migration and cell invasion. Conversely down-regulation of the FOXP3 increase cell migration and cell invasion(P <0.05).Conclusion:(1) Transcription factor FOXP3 in glioma cell 1ine U87 and LN229 is able to inhibite the pro1 iferation of tumor cells.(2) Transcription factor FOXP3 in glioma cell 1ine U87 and LN229 may through affect the expression of apoptotic proteins caspases-3 and caspases-7 to promote cell apoptosis of tumor cells.(3) Over-expression of FOXP3 in U87 cells and LN229 cells induced cell cycle arrest, resulting in U87, LN229 cell cycle arrest in G0 / G1 phase which may be a new strategy of targeted gene therapy glioma.(4) Over-expression of FOXP3 in U87 cells and LN229 cells inhibited cell migration and invasion, down-regulation of the FOXP3 increase cell migration and cell invasion.