Objective: NF-E2-related factor 2 (Nrf2) is a transcription factor that is related to tumor cell multidrug resistance and proliferation. Here we studied the involvement of Nrf2 in the migration and invasion of human U251 glioma cells.
Methods: Two kinds of plasmid, that is, pEGFP-Nrf2 and Si-Nrf2, were constructed and transfected to upregulate or downregulate the expression of Nrf2 in U251 glioma cell line. Blank vectors or random siRNA plasmid were used as negative control. Cells treated with lipofectamine only were set up as blank control. Protein and mRNA level of Nrf2 and matrix metalloproteinase 9 (MMP9) were investigated by reverse transcriptase-polymerase chain reaction and western blot after transfection. Wound healing assay and transwell assay were used to study migration and invasion of U251 after transfection. Gelatin zymography was performed to reveal the change of MMP9 activity after transfection.
Results: The mRNA and protein level of Nrf2 was upregulated in U251-pEGFP-Nrf2 while downregulated in U251-Si-Nrf2 48 hours after transfection. In the wound healing assay, there were more cells in group pEGFP-Nrf2 crossing the scratch line than in group Si-Nrf2. Furthermore, in transwell migration and invasion assay, there were more cells in group pEGFP-Nrf2 penetrating the membranes than in group Si-Nrf2. Then we investigated the change of MMP9 activity, mRNA, and protein levels after transfection. The results suggested that upregulation of Nrf2 led to an increase in MMP9 expression and activity whereas downregulation of Nrf2 led to a decrease in MMP9 expression and activity.
Conclusion: Nrf2 is involved in migration and invasion of U251 cells, which may be related to MMP9.
Keywords: ARE; DMEM; Dulbecco's modified Eagle medium; Glioma; Invasion; MMP9; Matrix metalloproteinase 9; Migration; NF-E2-related factor 2; Nrf2; RT-PCR; Reverse transcriptase–polymerase chain reaction; antioxidant response element.
Copyright © 2013 Elsevier Inc. All rights reserved.