Peer-Review ReportThe Role of Nrf2 in Migration and Invasion of Human Glioma Cell U251
Introduction
The transcription factor NF-E2-related factor 2 (Nrf2) is considered as a critical regulator of intracellular antioxidants and phase II detoxification enzymes by transcriptional upregulation of many antioxidant response element (ARE)-containing genes. It belongs to Keap1-Nrf2-ARE signaling pathway. In response to oxidative stress, Nrf2 is activated and dissociated from Keap1. After translocated from cytoplasm to nucleus, Nrf2 forms a heterodimer with Maf, which binds to the ARE sequence to upregulate many kinds of genes expression (21). The Nrf2 downstream genes include 1) intracellular redox-balancing proteins: glutamate cysteine ligase, glutathione peroxidase, and heme oxygenase-1; 2) phase II detoxifying >enzymes: glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase–1; and 3) transporters: multidrug resistance–associated protein (21). Recent studies have found more Nrf2 downstream genes that are involved in stress response, xenobiotics metabolism, the ubiquitin-mediated proteasomal degradation system, cell proliferation and so on (5, 20, 22, 36, 41). In normal tissue, Nrf2 is considered as an important transcript factor for cells to survive from many kinds of negative stimulus such as injury, ischemia, inflammation, hemorrhage, cancer, pulmonary fibrosis, acute pulmonary injury, and degeneration disease (6, 8, 18, 19, 36, 38, 42, 44). Interestingly, Nrf2 may play a dark role in tumor tissue. It has been verified that Nrf2 is upregulated in lung, head, and neck squamous cell carcinoma tissues (39, 40). Further investigation shows that overexpression of Nrf2 enhances tumor resistance to chemotherapeutic agents in some lung carcinoma, breast adenocarcinoma, and neuroblastoma cell lines (43).
Matrix metalloproteinase 9 (MMP9), which belongs to the ECM-degrading enzyme family, is a kind of gelatinase. MMP9 is involved in migration and invasion of tumor cells. It has been found that the transcription and activity levels of MMP9 are higher in glioma than in normal brain and such elevation is strongly correlated with the tumor grade (9). Our previous study (25) proved that MMP9 was upregulated in Nrf2 knockout mice than wild-type mice after spinal cord injury. This result suggests the possible relationship between Nrf2 and MMP9. In this study, we investigated the change of MMP9 level, migration, and invasion ability of human U251 glioma cells after up- or downregulating the expression of Nrf2.
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Cell Culture and Transient Transfection
Human U251 glioma cells were obtained from ATCC and cultured in Dulbecco's modified Eagle medium (DMEM; HyClone, Illinois, USA) with 10% fetal bovine serum (HyClone) at 37°C and 5% CO2 incubator. The primers for human Nrf2 cDNA were as follows: forward 5′-CCGCTCGAGATGATGGACTTGGAGCTGCC-3′, reverse 5′-GGGGTACCGTGTTTTTCTTAACATCTGGC-3′. Human Nrf2 cDNA was cloned into the cloning site of the vector pEGFP-N1 (GeneChem, Shanghai, China) using the standard recombinant DNA technique. The new plasmid
Transient Transfection Effect on Nrf2 mRNA and Protein Level
To validate the transient transfection effect of four plasmids mentioned above, we tested the mRNA and protein level of five cell groups 24, 48, 72 hours after transfection, respectively. RT-PCR and western blot results indicated that transfection efficiency was most predominant at 48 hours after transfection (data not shown). Transfection of pEGFP-Nrf2 enhanced the Nrf2 mRNA and protein level >38% and >130% as compared with transfection of pEGFP, respectively (P < 0.05). Si-Nrf2 transfection
Discussion
Nrf2 was first discovered by Moi in 1994 (27). Follow-up study showed that Nrf2 is a critical transcription factor in Keap1-Nrf2-ARE signaling pathway to resist oxidative stress. Nrf2 is considered as a “good” protein because many chemopreventive compounds have been found to be its inducer, such as sulforaphane (16), curcumin (1), epigallocatechin-3-gallate (29), resveratrol (4), wasabi (28), cafestol, kahweol (12), lycopene (3), and carnosol (37). All these compounds have been proven to be
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