proteins were extracted as described above. Aliquots of protein lysates were fractionated by SDS-PAGE, transferred to a nitrocel- lulose membrane (Whatman, Germany), and subjected to immuno- blotting analysis according to the manufacturer’s instruction. ECL Detection System (Thermo Scientific) was used for signal detection. ELISA assay for VEGF The protein levels of VEGF in the supernatant were measured using the Quantikine human VEGF ELISA kit (NeoBioscience, Shanghai, China) according to the manufacturer’s instruction. In brief, the cells were seeded in 6-well plates and cultured to 90% confluence, and then cells were switched to fresh medium. The supernatants were collected and the number of cells in each well was counted after 24 h. VEGF in the supernatant (100 ml) was determined, and normalized to the cell number. A serial dilution of human recombinant VEGF was included in each assay to obtain a standard curve. Cell proliferation assay To determine the effects of miR-128 on the cell proliferation, U87 and U251 cells (66104 cells) were seeded in a 6-well plate and cultured overnight. The cells were transiently transfected with pre- miR-128 and miR-SCR precursors, respectively; and cultured overnight. Then the cells were trypsinized and seeded at 2000 cells per well in a 96-well plate. The proliferation of the cells was measured using a Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instruction every 24 h after transfection. Three independent experiments were done in triplicate. Tumorigenesis in nude mice Male BALB/cA-nu nude mice (4-weeks-old) were purchased from Shanghai Experimental Animal Center (Chinese Academy of Sciences, Shanghai, China), and maintained in pathogen-free conditions. Eight mice were randomly divided into two groups. U87 cells stably expressing miR-128 were injected subcutaneously into both flanks of nude mice (56106 cells in 100 ml). U87 cells stably expressing miR-SCR were used as the negative control. Bi- dimensional tumor measurements were measured with calipers three times weekly. Tumor volumes were calculated according to the formula (width26length)/2. The mice were euthanized after 28 days, and tumors were weighed. Matrigel plug assay Male BALB/cA-nu nude mice (6-weeks-old) were purchased from Shanghai Experimental Animal Center (Chinese Academy of Sciences, Shanghai, China), and maintained in pathogen-free conditions. Eight mice were randomly divided into two groups. U87 cells stably expressing miR-128 were harvested and resus- pended in serum-free medium. Aliquots of the cells (26106 cells in 100 ml) were mixed with 200 ml of Matrigel. The mixture was immediately injected into both flanks of nude mice. U87 cells stably expressing miR-SCR in equal volumes of solvent were used as the control. On Day 11 after the implantation, the Matrigel plugs were trimmed out and used for the measurement of the hemoglobin content using Drabkin’s Reagent Kit (Sigma-Aldrich, St Louis, MO) according to the manufacturer’s instructions. The concentrations of hemoglobin were calculated based on a set of hemoglobin standards. Immunohistochemical analysis Tumors samples were fixed in Bouin’s solution and embedded in paraffin. Tumor sections at 5 mm were cut and deparaffinized, and antigen was retrieved by the heating using microwave for 15 min. MiR-128 Targets p70S6K1 After incubation with hydrogen peroxide, the sections were washed with 16PBS buffer for three times. The sections were blocked for 1 h with 5% Albumin Bovine serum in PBS buffer. Antibodies against HIF-1a (Bioworld, China), VEGF(Santa Cruz, CA, USA) and anti- Human CD31 (Abcam, USA) were used in a humid chamber at 4uC for 16 h. After washing three times, the sections were incubated with EnVision+, Peroxidase, Rabbit (Gene Tech, Shanghai, China) for 2 h. The protein signals were detected using 3,39-diaminobenzidi- ne(DAB) (Gene Tech, Shanghai, China). Different sections were prepared from three of the Matrigel plugs in every group, and the microvessels were counted in three different fields per section as follows: slides were first scanned under low power (6100) to determine three ‘‘hotspots’’ or areas with the maximum number of microvessels, then the positive-stained blood vessels in the selected areas were analyzed at 6400 magnification [17]. Statistical analysis All values in this study were presented as mean+SD. Statistical analysis was performed based on a Student’s t-test at the significance level of P,0.05. Spearman’s correlation analysis was used to determine the correlation of the expression levels of p70S6K1, miR-128, and the CD31-positive microvessel densities using SPSS software. Results MiR-128 expression was decreased in gliomas To determine the levels of miR-128 in glioma and normal brain tissues, total RNAs were extracted from normal brain tissues and glioma tissues at Grades II, III, and IV, and the expression levels of miR-128 were analyzed using RT-PCR and real time PCR. As shown in Figure 1A, the expression levels of miR-128 were down- regulated in glioma tissues when compared to the normal brain tissues. The levels of miR-128 expression were quantified using Figure 1. MiR-128 was down-regulated in human glioma tissues when compared to normal brain tissues. (A) Semi- quantative RT-PCR analysis of miR-128 levels in three normal brain tissues (N1–N3) and twelve glioma tissues (T1–T4 Grade II, T5–T8 Grade III, T9–T12 Grade IV). U6 RNA level was used as an internal control. (B) Relative miR-128 expression levels were analyzed by qRT-PCR in normal brain tissues and three grades of glioma tissues (Grade II, Grade III, Grade IV). U6 RNA level was used as an internal control. Double asterisks indicate significant difference when compared to normal brain tissues (P,0.01). doi:10.1371/journal.pone.0032709.g001 PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e32709