Cell lines and culture conditions
The human glioma cell lines U87MG, U251, U373 and the rat glioma cell line C6 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown in Dulbecco's modified Eagle's medium (DMEM, Cellgro Media tech)) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (10 IU/ml), and streptomycin (10 ug/ml). The cells were maintained at 37°C in a humidified air atmosphere at 5% CO2.
Serial selection for a sub-population of invasive cells using Boyden chambers
For selection of invasive cells, a suspension of 300,000 tumor cells/mL in serum-free DMEM (500 ul total volume) was plated in the upper chamber of a Boyden-type manifold, over a Matrigel-coated membrane (24-well insert; pore size, 8 um; BD Biosciences). DMEM medium containing 10% FBS in the lower chamber served as the chemoattractant. After incubation for 21 h at 37°C, those non-invading cells from the upper surface of the membrane were scrubbed off with cotton swabs. The cells that invaded to the bottom surface of the insert were trypsinized and seeded into flasks containing DMEM supplemented with 10% FBS. These cells were propagated and went through the above selection procedure twice more. The cells that were collected after the third selection procedure were propagated resulting in the invasive sub-population cell line termed IM3.
Boyden chamber invasion assay
For migration assays, a suspension of 300,000 tumor cells/ml in serum-free DMEM (500 ul total volume) was plated in the upper chamber of a Matrigel-coated membrane insert (24-well insert; pore size, 8 um; BD Biosciences). DMEM medium containing 10% FBS in the lower chamber served as the chemoattractant. The cells were incubated at 37°C for various times depending on our laboratory experience regarding their relative speeds of invasion (18 hrs for U87 and U251; 36 hrs for U373; 17 hrs for C6). The non-invading cells were removed with cotton swabs. Those cells that had migrated to the lower side of the membrane were fixed and stained with hematoxylin. The invading cells on quadruplicate membranes were quantitated by counting across a diameter of each memebrane under 40X microscopic magnification.
Cell attachment assay
For the cell attachment assay, wells of a 24-well tissue culture plate were coated with Matrigel Basement Membrane Matrix (BD Biosciences) diluted 1:10 with serum-free medium for 2 h at room temperature. The unbound material was aspirated and the wells were rinsed with serum-free medium. Cells were detached with trypsin/EDTA and rinsed in serum free medium. Approximately 20,000 cells/ml in 10% DMEM were plated into the wells, and the plate was incubated at 37°C for 4 h. Wells were gently washed with PBS and the attached cells were fixed in 4% paraformaldehyde and stained with hematoxylin QS (Vector Laboratories) for 2 min. Wells were washed again in PBS and the stained cells were counted.
For each cell line, parental and IM3 cells were plated at a density of 5000 cells per well, each in 8 wells of 96-well plates containing 10% FBS/DMEM and maintained at 37°C. The rate of cell proliferation was determined using CellTiter 96 AQueous
One Solution (Promega, Madison, WI) at 4, 24, 48, 72 and 96 hours after plating. An MTS assay was performed by following the protocol previously reported [8
]. Eight media only containing wells were used to normalize the absorbance values of the wells containing cells. Growth curves were generated with normalized absorbance compared to the 4-hour data, at 24, 36, 48, and 72 hours.
Total cellular RNA was extracted using the mirVana miRNA Isolation kit (Ambion) per manufacturer's instructions for total RNA isolation. The RNA concentration was determined by spectrophotometric absorbance at 260 nm, and the quality of the RNA was determined by Agilent 2100 Bioanlayzer instrument (Agilent Technologies, Santa Clara, CA). All RNA samples showed high quality (RNA integrity number (RIN) > 9.0) and were without RNA degradation or DNA contamination.
Real-time quantitative RT-PCR analysis
qRT-PCR was performed in triplicate by using LNA™ PCR primer sets (miR-143, -145 and U6), SYBR®Green PCR kit, and the ViiA™ 7 real-time PCR system (Exiqon). Total RNA extracted from U87, U251, U373, and their corresponding invasive subpopulations, was used for cDNA synthesis (Exiqon). The PCR reaction was started with cDNA (140 ng/μl) in 25 μl (total) assay mix. The PCR cycle settings were those recommended by Exiqon: 95°C, 10 min; 40 amplification cycle at 95°C (10 s) and 65°C (1 min), 1.6°C/s ramp-rate.
Knockdown miRNA transfection
Parental and IM-3 cells from each of three cell lines (U87, U251, U373) cells were transfected with either fluorescein-labeled locked nucleic acid (LNA) antagomirs targeting miR-143 and miR-145 (Exiqon, Vedbaek, Denmark) at a final concentration of 50 nM (each probe) using Lipofectamine 2000 per manufacturer's instructions (Invitrogen, Carlsbad, CA). Control cells were transfected with an un-targeted antagomir at a final concentration of 100 nM. Transfection medium was changed 8 hours after transfection and replaced with Optimem medium (Invitrogen Carlsbad, CA) for overnight recovery. The efficiency of the transfection was assessed with fluorescence microscope. A Double knockdown transfection of U87 and U87 IM3 cells using fluorescein-labeled miR-143 and miR-145 knockdown probe at final concentration of 50 nM each and control LNA knockdown probe at final concentration of 100 nM was performed using Lipofectamine 2000 per manufacturer's instructions. Once again, culture medium was changed 8 hours after transfection and replaced with Optimem medium (Invitrogen Carlsbad, CA) for overnight recovery and the efficiency of the transfection was assessed with fluorescence microscopy. Antisense micro RNAs (antimir-143 and -145) for knockdown were purchased from Exiqon.
Boyden chamber migration assay of knockdown miRNA Transfected cells
A suspension of 300,000 cells/mL in serum-free medium of parental and IM3 cells transfected with LNA antagomirs (against miR-143 and miR-145 or a control LNA antagomir) was plated in the upper chamber of a Boyden manifold over a Matrigel-coated membrane (24-well insert; pore size, 8 um; BD Biosciences). DMEM medium containing 10% FBS in the lower chamber served as the chemoattractant. The cells were incubated for 17-36 hours at 37°C. The non-invading cells were removed with cotton swabs. Those cells that had migrated to the lower side of the membrane were fixed and stained with DAPI. The invading cells of quadruplicate membranes were counted under the microscope at 40× magnification.
In situ detection of miRNAs
In situ detection of miRNAs was performed on 8-10-um frozen tissue sections from xenografts of human GBM tumors in mouse brain. Sections were fixed using fresh ice-cold 4% paraformaldehyde for 1 h, acetylated in acetic anhydride/triethanolamine, and prehybridized in hybridization solution (50% formamide, 5X SSC, 0.5 mg/mL yeast tRNA, 1X Denhardt's solution) at 25°C below the predicted Tm value of the LNA probe for 30 min. Probes (4 pmol; fluorescein isothiocyanate (FITC)-labeled LNA-modified oligonucleotide; Exiqon) complementary to the mature miRNA (miR-143 and miR-145) were hybridized to the sections for 2 h at 25°C lower than predicted Tm value of the LNA probe. Post-hybridizations washes were performed in 0.5X SSC at 8°C above the hybridization temperature and the in situ hybridization signal was detected by incubation with horseradish peroxidase (HRP)-conjugated anti-FITC. The signal was then amplified using FITC-conjugated tyramide (Tyramide Signal Amplification Plus Kit, Fluorescein, Perkin-Elmer) according to the manufacturer's instructions. Slides were mounted in Vectashield Hard Set mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Vectashield mounting medium with DAPI, Vector Laboratories) and analyzed with an Olympus CKX41 microscope equipped with a CCD camera and Olympus software.
Data are presented as mean +/- standard deviation. For parental and IM-3 comparisons, the Student's t test (two-tailed) was used to determine statistical significance. A student's t test with a value of P < 0.05 was considered significant.