Cells and cell treatments
Glioma cell lines LN229 and A172 were grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 100 units penicillin and 100 units streptomycin at 37°C in 5% CO2 atmosphere in a humidified incubator. The MMP inhibitor and NF-kappaB activation inhibitor II JSH-23 (EMD, La Jolla, CA) compounds were dissolved in dimethyl sulfoxide (DMSO) and used, respectively, at 50μM and 30μM. Treatment of cells with the MMP inhibitor was performed for indicated time lengths, and JSH-23 was used to treat cells for 11 h.
Vectors and retroviral infection
pMSCV/Bmi-1 overexpressing human Bmi-1 was constructed as previously described . To silence endogenous Bmi-1 expression, Bmi-1 RNA interference (RNAi) sequence (5’-ATGAAGAGAAGAAGGGATT-3’, synthesized by Invitrogen) was cloned into retroviral transfer vector pSuper-retro-puro. As described previously , retroviral particles were produced by cotransfection with pSuper-retro-Bmi-1-shRNA and PIK packaging plasmid into 293 T cells and collected 24 to 48 hrs after transfection to infect glioma cells. Stable cell lines expressing Bmi-1 or with Bmi-1 silenced were selected by treatment with 0.5 μg/ml puromycin for 10 days, beginning from 48 hours after infection . After selection for 10–14 days, the cell lysates prepared from the pooled population of cells in the sampling buffer were fractionated on SDS-PAGE for immunoblotting detection of Bmi-1 protein level.
Real-time RT-PCR and data analysis
Total cellular RNA was extracted using the Trizol reagent (Invitrogen) according to the manufacturer’s instruction. Two micrograms of RNA from each sample were used for cDNA synthesis primed with random hexamers. For PCR amplification of cDNA, an initial amplification using gene-specific primers was done with a denaturation step at 95°C for 10 minutes, followed by 28 cycles of denaturation at 95°C for 60 seconds, primer annealing at 58°C for 30 seconds, and primer extension at 72°C for 30 seconds. At completion of the cycling, a final extension at 72°C for 5 minutes was done before the reaction was terminated. Expression levels of genes were normalized to housekeeping gene GAPDH as the control. PCR primers were designed by employing the Primer Express version 2.0 software (Applied Biosystems, Foster City, CA). The Primers was shown as following: MMP9-up: ACGACGTCTTCCAGTACCGA; MMP9-dn: TTGGTCCACCTGGTTCAACT; CCND1-up: AACTACCTGGACCGCTTCCT; CCND1-dn: CCACTTGAGCTTGTTC ACCA; Bcl-xL-up: ATTGGTGAGTCGGATCGCAGC; Bcl-xL-dn: AGAGAAGGGGG TGGGAGGGTA; TNF alpha-up: CCAGGCAGTCAGATCATCTTCTC; TNF alpha-dn: AGCTGGTTATCTCTCAGCTCCAC; VEGFC-up: GTGTCCAGTGTAGATGAACTC; VEGFC-dn: ATCTG TAGACGGACACACATG; MYC-up: TTCGGGTAGTGGAAAACCAG; MYC-dn: CAGCAGCTCGAATTTCTTCC; GAPDH-up: GACTCATGACCACAGTCCATGC; GAPDH-dn: AGAGGCAGGGATGATGTTCTG.
Western blotting analysis
Western blotting analysis was performed according to standard methods as previously described .The membrane was probed with a 1:500-diluted rabbit anti-human Bmi-1 antibody (Cell Signaling, Danvers, MA). The membranes were stripped and re-probed with a mouse anti-β-actin monoclonal antibody (1:1,000; Sigma, Saint Louis, MI) as a loading control.
Wound healing assay
Cells were seeded on six-well plates with DMEM containing 10% FBS and grown to confluence. The cells were scratched with a sterile 200μL pipette tip to create artificial wounds. At 0 and 24 hr after wounding, respectively, phase-contrast images of the wound healing process were photographed digitally using an inverted Olympus IX50 microscope with 10× objective lens. Eight images per treatment were analyzed to determine averaging parameters of positioning of the migrating cells at the wound edges by digitally drawing lines using the Image-Pro Plus software (Media Cybernetics).
Transwell migration assay and Transwell matrix penetration assay
Cells (1 × 104) to be tested were plated on the top side of the polycarbonate Transwell filter without (for Transwell migration assay) or with Matrigel coating (for Transwell matrix penetration assay) in the upper chamber of the BioCoatTM Invasion Chambers (BD, Bedford, MA) and incubated at 37°C for 22 hrs, followed by removal of cells inside the upper chamber with cotton swabs. Migrated and invaded cells on the membrane bottom-surface were fixed in 1% paraformaldehyde, stained with hematoxylin, and counted (Ten random 200× fields per well). Cell counts were expressed as the mean number of cells per field of view. Three independent experiments were performed and the data are presented as mean ± standard deviation (SD).
3-D spheroid invasion assay
The Matrigel matrix (BD Biosciences, San Jose, CA) was used in 3-D spheroid invasion assay, which displays morphologies typical of highly aggressive invasiveness presenting more outward projections (Invadopodia or invasive feet) [29–33]. Indicated cells (1 × 104) were trypsinized and seeded in 24-well plates coated with Matrigel (2%, BD Biosciences), and medium was changed every other day. Pictures were taken under microscope at 2-day intervals for 2–3 weeks.
Immunohistochemical analysis (IHC)
IHC was performed according to standard methods as previously described . Sections were IHC analyzed using anti-Bmi-1, anti-MMP-9 and anti-NF-kappaB antibodies (Cell signaling, Danvers, MA,). Images were captured using the AxioVision Rel.4.6 computerized image analysis system (Carl Zeiss Co. Ltd., Jena).
Cells (1.5 × 104) were seeded in triplicates in 24-well plates and allowed to settle for 24 hrs. One hundred nanograms of luciferase reporter plasmid containing fragments of the MMP-9 promoter with serial deletions, pNF-kappaB-luc plasmid, or the control-luciferase plasmid, in combination with 1 ng of pRL-TK renilla plasmid (Promega,Madison, WI), were transfected into glioma cells using the Lipofectamine 2000 reagent (Invitrogen, Co., Carlsbad, CA) according to a protocol provided by the manufacturer. Luciferase and renilla signals were measured at 48 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI) according to the manufacturer’s instruction. Three independent experiments were performed and the data are presented as mean ± SD.
Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed using a commercial kit according to the manufacturer’s manual (Keygentec, Shanghai). Briefly, 100 μl of diluted standard and tested samples, namely, Bmi-1-overexpressing or -silencing cells, and the vector control cells, including a negative control, were added to the ELISA plate and incubated at 36°C for 90 min. After the unbound samples were washed off by DI water and PBS-Triton, specific antibody (anti-MMP-9, anti-MMP-2, or anti-MMP-7) was incubated with the plate at 36°C for 60 min. After further washing steps, 100 μl of second antibody was added and incubated for 60 min. Subsequently, the substrate was added and incubated at RT for 60 min before the reaction was stopped, followed by the results-reading with a microplate reader. Colorimetric measurement was recorded as OD450 readings.
Gelatin zymography assay
Cells were seeded in 48-well culture plates at a density of 3 × 104/well and incubated for 24 h before the medium was replaced with serum-free medium (Invitrogen, Carlsbad, CA), followed by collection of conditioned medium and quantification for protein contents. Samples containing equal amounts of protein (1 μg/μl) mixed with 4 × sampling buffer (3:1) were run on 9% polyacrylamide gels containing 0.2% gelatin (Sigma, Saint Louis, MI). After electrophoresis, the gel was washed twice in wash buffer (2.5% Triton X-100, 50 mM Tris–HCl, 1 μM ZnCl2, pH 7.6) for 45 min/each time, followed by two rinses with the wash buffer (without Triton X-100) and subsequent incubation at 37°C in 50 mM Tris–HCl (pH7.6), 5 mM CaCl2, 1 μM ZnCl2 and 0.02% Brij-35 for 16 h. The gels were stained with 0.1% Coomassie brilliant blue R-250 and then de-stained with de-staining solution (40% methanol, 10% acetic acid in distilled water). A protein marker was used to measure the molecular weights of proteins in SDS-PAGE before the staining and washing step of the Zymography Assay. Meanwhile, the MMP-9 recombinant protein was also used as the standard to confirm the band of MMP-9.
Tissue specimens and patient information
Paraffin-embedded, archived glioma specimens were histopathologically diagnosed at the First Affiliated Hospital of Sun Yat-sen University from 2000 to 2005. The clinical information is described in Additional file 1: Table S1. The use of the clinical specimens was approved by the local Institutional Review Board, the Ethical Committee of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China, and conformed to the ethical guidelines of the Helsinki Declaration.
Statistical analyses were performed using the SPSS 11.0 statistical software package. Data represent mean ± SEM. P values of 0.05 or less were considered statistically significant.