Netrin-1 promotes glioma growth by activating NF-κB via UNC5A

Gliomas, a common type of brain tumor, are characterized by aggressive infiltration, making it difficultly to cure by surgery. Netrin-1, an extracellular guidance cue critical for neuronal axon path-finding, has been reported to play an important role in cell invasion and migration in several types of cancers. However, the role of netrin-1 in glioma remains largely unknown. Here, we provide evidence suggested that Netrin-1 has a critical role in glioma growth. We found that netrin-1 was significantly increased in glioma samples and positively correlated with cell proliferation, tumor grade and malignancy. Netrin-1 knockdown reduced cell proliferation and attenuated tumor growth in a xenograft mouse model. Further studies found that netrin-1 induced NF-κB p65ser536 phosphorylation and c-Myc expression in vitro and in vivo. Interestingly, activation of NF-κB by netrin-1 was dependent on UNC5A receptor, because suppression of UNC5A significantly inhibited NF-κB p65ser536 phosphorylation, c-Myc up-regulation and reduced cell proliferation. Taken together, these results suggested netrin-1 promotes glioma cell proliferation by activating NF-κB signaling via UNC5A, netrin-1 may be a potential therapeutic target for the treatment of glioma.

Immunocytochemical (ICC) and immunohistochemical (IHC) staining. The paraffinembedded tissue sections used for ICC were subjected to the removal of paraffin with xylene, followed by rehydration with ethanol. For antigen retrieval, the sections were placed in citrate buffer (pH=6.0) at a temperature of 120°C and a pressure of 0.12 MPa for 2 min. A similar procedure was followed for adherent cells, except that neither removal of paraffin nor antigen retrieval was required. Briefly, the sections or the adherent cells were rinsed three times in PBS, blocked with 2% BAS in PBS at room temperature for 2 h, incubated with the primary antibody at 4°C overnight and then incubated with the appropriate fluorescent secondary antibodies for 1 h at room temperature. The nuclei were stained with 1 mg/mL DAPI (1:200) for 6 min.
For the paraffin-embedded tissues used for IHC, a procedure similar to that performed for ICC was carried out. After antigen retrieval, the sections were washed with PBS three times, using 3% H2O2 for 10 min to block endogenous peroxidase activity. After blocking, the sections were incubated with the primary antibody, followed by incubation with the secondary antibody according to the UltraSensitive TM Cell proliferation assay. Cell growth rates were assessed by the MTT and CCK-8 assays. For the MTT assay, 2 × 10 3 U87MG cells, 2 × 10 3 SHG44 cells, and 3 × 10 3 U251 cells were seeded onto 96-well plates in triplicate. After incubation for the indicated length of time, 20 μl of MTT (5 mg/ml) was added to each well, and the cells were incubated for 4 h at 37°C before removal of the culture medium. The supernatant was removed, and then 100 µl of DMSO was added, and plates were placed on a shaker for 15 min in the dark at room temperature. Cell viability was determined by measuring the absorbance at 490 nm. For the CCK-8 assay, cells were seeded onto 96-well plates. After incubation for the indicated length of time, 10 µl of CCK-8 solution was added to each well, and the cells were incubated for 2 h at 37°C. Then, cell viability was determined by measuring the absorbance at 450 nm.
BrdU assay. In brief, 6 h after the addition of BrdU (59-14-3, Sigma-Aldrich, St. Louis, MO), the media were removed, and the cells were fixed with 4% PFA for 10 min. Then, the cells were subjected to three 5 min washes in PBS. Next, 2 M HCl was added to the cells, and the cells were incubated for 30 min at room temperature. The HCl was removed by aspiration. Then, 0.1 M boric acid was added, and the cells were incubated for 2 min. Next, the cells were subjected to three 5-min washes. Then, BSA was added, and the cells were incubated for 2 h at room temperature. After that, the BrdU antibody was added, and the cells were incubated overnight at 4°C. After incubation, the cells were washed with PBST 6 times for 5 min each, followed by incubation with the appropriate fluorescent secondary antibodies for 1 h at room temperature. The nuclei were stained with 1 mg/mL DAPI (1:200) for 6 min.
Soft agar colony formation assay. The 0.6% low melting point (LMP) agarose was prepared in deionized H2O, heated, and cooled. Equal amounts of agarose and DMEM containing 20% FBS were combined. A total of 2 ml of this solution was added to the wells of the 6-well plates, and the plates were incubated for 30 min at 4°C to allow the agar to solidify. This formed the bottom layer of the soft agar.
The cells were trypsinized and resuspended at concentrations of 3 × 10 3 cells per well in DMEM containing 0.3% agarose and 10% FBS. The plates were incubated for 30 min at 4°C to allow the agar to solidify. Once the agar was solid at room temperature, the plates were incubated at 37°C for 16 days.
Colonies were counted using a dissecting microscope, and counts were calculated based on three replicate wells for each condition.
Flow Cytometry. Cells were collected by centrifugation, and the supernatant was aspirated. The cells were stained with 50 μg/ml propidium iodide and Annexin V-fluorescein isothiocyanate (Kaiji) following the manufacturer's instructions. The data were analyzed using a flow cytometer.

Lentiviral packaging and infection of the target cells. The scrambled shRNA oligo
TTCTCCGAACGTGTCACGT, which has no specific targets in humans, was used as a negative control.
The shRNA target sequences are listed below. The concentrated virus was used to infect U251, SHG44 and U87MG cells.

Transwell migration and invasion assays.
In vitro cell migration and invasion assays were performed as previously described using Transwell chambers (24-well inserts, Corning 8-μm pore size; Corning Costar, Corning, NY, USA). For the migration assay, 2 × 10 4 cells in 300 µl of DMEM were added to the top chambers, while 700 µl of complete medium was added to the lower chambers. The cells were then incubated for 20 h in a humidified cell culture incubator. After incubation, the cells were washed with PBS three times, fixed with 4% PFA and then stained with 0.1% Crystal Violet. The cells that had not migrated were removed from the upper face of the filters using cotton swabs. Images of five random fields were captured from each membrane, and the number of migratory cells were counted.
Similar inserts coated with Matrigel (BD Biosciences) were used to determine invasive potential, and 5x10 4 cells were used in the invasion assay. Luciferase-expressing U251 cells (4 × 10 5 ) infected with a virus coding for the scramble shRNA or Netrin-1 shRNAs were injected into the right striatum of the brain in five randomly selected mice per group. The injection site for the mice was determined as follows using the position of the bregma: 1 mm anterior, 2 mm lateral and 3 mm intraparenchymal (from the dura). The tumor cells were slowly injected over 7 min. All procedures were carried out under sterile conditions. After injection, all mice were monitored daily. In a few cases, the mice died a few hours after transplantation, and these mice were excluded from the analysis. In the end, the total number of mice in each group was at least four.
The tumor-bearing mice were imaged in vivo with bioluminescence on the 16 th day after injection and sacrificed on the 27 th day when the mice exhibited difficulty eating, cachexia, or action disorder. The brains of the mice were fixed with 4% poly-formaldehyde and processed for paraffin embedding. Then, 5-µm tissue sections were stained by ICC or IHC.

Supplementary Figure
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