Eagle's minimum essential medium (MEM), Dulbecco's modified essential medium (DMEM) with 4.5 g/L glucose, DMEM with 1 g/L glucose, 0.15% sodium bicarbonate, 1 mmol/L sodium pyruvate, 0.1 mol/L nonessential amino acids and HEPES buffer were purchased from Cellgro Mediatech (Washington, DC). Improved MEM Zinc Option, neurobasal media, N2, B27, penicillin-streptomycin, Trizol, Oligofectamine and pcDNA3.1/Zeo vector were purchased from Invitrogen (Carlsbad, CA). Human recombinant bFGF and EGF were purchased from R&D Systems (Minneapolis, MN). Fetal bovine serum (FBS) was purchased from GEMINI Bio-Products (West Sacramento, CA). Pre-miR-34a or control pre-miR (miR-con), and pmiR-REPORT vector were purchased from Ambion (Austin, Texas). pGL3-promoter plasmids and Luciferase System Kit were purchased from Promega Corp (Madison, WI). Crystal violet was purchased from Sigma (Saint Louis, Missouri). Fugene 6 reagent was purchased from Roche (Indianapolis, IN). Bio-Rad's iScript cDNA Synthesis Kit was purchased from Bio-Rad Laboratories (Hercules, CA). Taqman MicroRNA Reverse Transcription Kit, MultiScribe reverse transcriptase and a human 18S rRNA Taqman probe were purchased from Applied Biosystems (Foster City, CA). Propidium iodide, Annexin V-PE and 7AAD were purchased from BD Pharmingen (San Diego, CA).
U87 human glioblastoma cells were grown in Eagle's MEM supplemented with 10% FBS, 0.15% sodium bicarbonate, 1 mmol/L sodium pyruvate, 0.1 mol/L nonessential amino acids, and 500 μg/mL penicillin-streptomycin. A172 human glioblastoma cells and LN-Z308 cells (a kind gift from Dr. Erwin Van Meir, Emory University) were grown in DMEM with 4.5 g/L glucose and 10% FBS. U373 and T98G human glioblastoma cells were grown in DMEM with 1 g/L glucose supplemented with HEPES buffer and 10% fetal calf serum. DAOY human medulloblastoma cells were grown in Improved MEM Zinc Option supplemented with 10% FBS. Glioma stem cells 0308 (a kind gift from Dr. Howard Fine, NIH) were grown in Neurobasal Media, N2 and B27 supplements (0.5 × each) and human recombinant bFGF and EGF (50 ng/ml each). Immortalized human astrocytes (a kind gift of Dr. Russ Pieper, UCSF) were grown in DMEM with 4.5 g/L glucose supplemented with 10% FBS. All cells were grown at 37 °C in 5% CO2-95% O2.
The pcDNA-c-Met, pcDNA-Notch1 and pcDNA-Notch2 plasmids were constructed via respective insertions of the full-length human c-Met, Notch1 and Notch2 cDNAs that do not contain the 3′UTR regions into the pcDNA3.1/Zeo vector. The Notch-1 3′UTR reporter plasmid was constructed via insertion of Notch-1 3′UTR into the pGL3-promoter plasmid. The Notch-2 3′UTR reporter plasmid was constructed via insertion of Notch-2 3′UTR into the pmiR-REPORT vector. The c-Met 3′-UTR reporter plasmid was a kind gift of Dr. Lin He (Cold Spring Harbor Laboratory) (25
). The miR-34a reporter plasmid was constructed via insertion of two copies of full site complementary to miR-34a into the pGL3-promoter plasmid. The p53 and mutant-p53 expression plasmids were kind gifts of Dr. Bert Vogelstein (Johns Hopkins University).
Quantitative Real-Time RT-PCR
Patient glioblastoma specimens and normal brain samples were obtained from University of Virginia Health System under an approved IRB protocol. Total RNA was extracted with Trizol according to the manufacturer's instructions. For endogenous controls, cDNA was synthesized using Bio-Rad's iScript cDNA Synthesis Kit. For microRNA expression analysis, 10 ng total RNA was used along with miR-34a–specific primers supplied with miR-34a Taqman MicroRNA Assay. cDNA was synthesized using Taqman MicroRNA Reverse Transcription kit and quantitative real-time PCR analysis was performed using the 7500 Real-Time PCR System. A human 18S rRNA Taqman probe was used as endogenous control. For c-Met expression analysis 50 ng total RNA was used and cDNA was synthesized using Multiscribe reverse transcriptase and c-Met expression was analyzed using c-Met specific primers. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were used as endogenous controls.
Transfections of microRNAs were performed using Oligofectamine and 10 nM pre-miR-34a or 10 nM pre-miR-con according to the manufacturer's instructions. Plasmid transfections were performed with Fugene 6 reagent according to the manufacturer's instructions.
To determine if miR-34 can bind to c-Met, Notch-1 or Notch-2 3′-UTR, brain tumor cells or stem cells were transfected with pre-miR-34a or pre-miR-con for 24 hrs and subsequently transfected with either 3′UTR-control, 3′UTR-Met, 3′UTR-Notch-1, or 3′UTR-Notch-2 in addition to control cytomegalovirus-β-galactosidase reporter plasmids for 48 hrs. To determine if wild-type p53 but not mutant p53 activates miR-34a, p53-null LN-Z308 cells were transfected with pre-miR-34a or pre-miR-con reporter plasmids for 6 hrs prior to transfection with either wild-type p53, mutant p53 (R273H) or control vectors for 48 hrs. Luciferase assays were performed using the Luciferase System Kit and luminescence was measured on a Promega GloMax 20/20 luminometer and normalized as described previously (28
Immunoblotting was performed as previously described using antibodies specific for c-Met and CDK6 (Cell Signaling Technologies, Danvers, MA), Notch-1, Notch-2 (Santa Cruz Biotechnologies, Santa Cruz, CA). All blots were stripped and re-probed with β-actin or α-tubulin antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA) as loading controls.
Cell proliferation assays
U87, A172 and DAOY cells (30,000/well) were transfected with pre-miR-34a or pre-miR-con as described above. After 72 hrs, the cells were collected every day for five subsequent days and counted with a hemocytometer.
Propidium Iodide Flow Cytometry
The effects of miR-34a expression on cell cycle progression were assessed using propidium iodide (PI) flow cytometry as previously described 28, 29. Briefly, U87 and A172 cells or astrocytes were transfected with pre-miR-34a or pre-miR-con for 72 hrs. The cells were washed with PBS, harvested and fixed in 70% (v/v) ethanol. The cells were then treated with 20 μg of DNase-free RNase and stained with propidium iodide. Cell samples were analyzed on a FACscan (Becton-Dickinson, Fullerton, CA) and G0/G1, S and G2/M fractions were determined.
Annexin V-PE and 7AAD flow cytometry
The effects of miR-34a expression on cell death were assessed by Annexin V-PE and 7AAD flow cytometry as previously described 1. Briefly, A172, 0308, DAOY and astrocytes were transfected with pre-miR-34a or pre-miR-con for 96 hrs. The cells were harvested and stained with Annexin V-PE and 7AAD according to the manufacturer's instructions. Cell samples were analyzed on a FACsan and apoptotic fractions were determined.
Cell invasion assays
The effects of miR-34a expression on cell invasion were assessed using a transwell invasion assay as previously described (28
). Briefly, A172 cells were transfected with pre-miR-34a or pre-miR-con for 72 hrs. The transfected cells (1×105
) were resuspended in 300 μL 0.1% FBS medium and placed in the upper chamber of the wells. Six hundred μL 10% FBS medium were placed in the lower chamber. After incubation for 6 hrs at 37°C in 5% CO2, the cells on the upper membrane surface were mechanically removed. Cells that had migrated to the lower side of the collagen IV-coated membrane were fixed and stained with 0.1% crystal violet. Migrated cells were counted under a microscope in five randomly chosen fields and photographs were taken.
c-Met and Notch rescue experiments
To determine if c-Met or Notch can rescue miR-34a-induced cell cycle arrest or cell death, the effects of miR-34a on cell cycle and apoptosis were tested in the setting of forced expression of plasmids encoding either c-Met or Notch1 and Notch2 transcripts lacking the respective 3′-UTR regions and that therefore cannot be inhibited by miR-34a. The plasmids were transfected in U87 or 0308 cells for 6 hrs prior to transfection with pre-miR-34a or pre-miR-con for 48 hrs. The cells were collected and analyzed for cell cycle with propidium iodide flow cytometry or for cell death with Annexin V-PE/7AAD flow cytometry as described above. c-Met and Notch expression changes were verified by immunoblotting.
Tumor formation in vivo
The effects of miR-34a on in vivo tumor growth were tested in an intracranial glioma xenograft model. U87 cells were transfected with pre-miR-34a or pre-miR-con for 24 hrs. Transfected cells (3 × 105) were stereotactically implanted into the striata of immunodeficient mice (n=10). The animals were sacrificed after 4 weeks of tumor implantation. The brains were removed, sectioned, and stained with H&E. Maximal tumor cross-sectional areas were measured by computer-assisted image analysis and tumor volumes were calculated.
When appropriate, two group comparisons were analyzed with a t-test and multiple group comparisons were analyzed with a Dunnett test and p values were calculated. P < 0.05 was considered significant and symbolized by an asterisk in the graphs.