miR-128. In addition, miR-128 overexpression suppressed cell proliferation and attenuated tumor growth and angiogenesis in vivo. These results give novel insights into our understanding the role and mechanism of miR-128 in glioma pathoetiology, and provide a potential therapeutic strategy for glioma treatment in the future. Materials and Methods Human tissue samples All human normal brain and glioma tissue samples were obtained from the Department of Neurosurgery, the first affiliated hospital of Nanjing Medical University. This study procedure was approved by The Institutional Review Board at the hospital. All participants provided written informed consent. Tissue samples were collected during surgery. For each sample, the major portion of tissue was frozen immediately in liquid nitrogen for molecular analysis, and the remaining tissue was fixed in paraformaldehyde for histological examination. All samples were histologically classified and graded according to WHO guidelines by clinical pathologist, and were prepared for cases in the institute biorepository, and classified and selected based on diagnosis. No information regulated by The Health Insurance Portability and Accountability Act was included in the study, which qualifies for the status of USA NIH Exemption # 4. Cell culture and reagents Human glioma cell lines, U87 and U251 were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 units of penicillin/ml, and 100 ng of streptomycin/ml. HEK-293 cells were cultured in DMEM medium supplemented with 10% FBS, 100 units of penicillin/ml, 100 ng of streptomy- cin/ml., and 2 mmol/ml glutamine. All cells were incubated at 37uC supplemented with 5% CO2. Antibodies against HIF-1a and HIF-1b, and the growth factor-reduced Matrigel were from BD Biosciences (Bedford, MA, USA). The antibodies against phospho- p70S6K1 (Thr389), p70S6K1 were from Cell Signaling Technol- ogy (Beverly, MA, USA). Monoclonal antibody against b-actin was from Sigma. Antibodies against GAPDH were from Kangcheng Company (Shanghai, China). Lentiviruses packaging and stable cell lines The hsa-miR-128 and hsa-miR-scrambled control (miR-SCR) lentiviral constructs were purchased from Thermo Fisher Scientific (Huntsville, AL, USA). To establish stable cells expressing miR- 128 and miR-SCR, lentiviruses were packaged using Lentiviral Packaging System according to the manufacturer’s instructions (Thermo Fisher Scientific). The lentiviral supernatant obtained from HEK-293 cells was used to transduce U87 and U251 cells. Stable cell lines were selected by puromycin. RNA extraction, reverse transcription PCR and quantitative real time- PCR Total RNAs were extracted from cells with Trizol (Invitrogen, CA, USA). One microgram of RNAs was used for cDNA synthesis using oligo(dT)15 and M-MLV reverse transcriptase. Aliquots of cDNAs were amplified with primers for VEGF and GAPDH. The primers are as follows: VEGF forward primer, 59-TCGGGC- CTCCGAAACCATGA-39; VEGF reverse primer, 59-CCTGG- TGAGAGATCTGGTTC-39; GAPDH forward primer, 59-CC- ACCCATGGCAAATTCCATGGCA-39; GAPDH reverse prim- er, 59-TCTAGACGGCAGGTCAGGTCCACC-39. VEGF and GAPDH were amplified by PCR for 30 cycles with each cycle at 95uC for 1 min, 59uC for 30 s, and 72uC for 1 min. The relative MiR-128 Targets p70S6K1 VEGF mRNA levels were normalized to those of GAPDH mRNA levels using Quality One analysis software (Bio-Rad, USA). To analyze miR-128 expression levels, RNAs were extracted from tissues. The stem-loop RT-PCR assay was used to quantify the miRNAs expression levels as described previously [24,25]. The RT-PCR primers were as following: miR-128 RT primer: 59-C- TCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGA- AAAGAGA-39. miR-128 PCR primers: sense: 59-ACACTCCA- GCTGGGTCACAGTGAACCGGTC-39; anti-sense: 59- TGGT- GTCGTGGAGTCG-39. U6 RT primer: 59-TGGTGTCGTG- GAGTCG. U6 PCR primers: Sense: 59-CTCGCTTCGGCAG- CACA; anti-sense: 59-AACGCTTCACGAATTTGCGT. PCR products were separated on 2.0% agarose gels stained with ethidium bromide, and visualized under UV light. RNA input was normalized to the level of human U6 snRNAs. qRT-PCR was performed using SYBR Premix DimerEraser (Takara, Dalian, China) on a 7900HT system. MiR-128 and U6 PCR primers were the same as described previously. The expression levels of miR- 128 were normalized with reference to expression levels of U6 snRNA, and fold changes were calculated by relative quantifica- tion (22DDCt) [26]. Vector construction pRK7-HA-S6K1 expression vector was as previously described [27]. The p70S6K1 39-UTR sequence was amplified from human cDNAs by PCR using the following primers: p70S6K1 forward primer, 59-CGCGGATCCGTAAATGGCTTGTGATACTCT- TGA-39; p70S6K1 reverse primer, 59-CGCCTCGAGATAG- GTGTGTGCTTTCTGTCTGTG-39. For its mutagenesis, the sequence complementary to the binding site of miR-128 in its 39UTR (CACTGTGA) was replaced by CAGTCTCA. The wild- type and mutated 39-UTRs of p70S6K1 were cloned into pMIR- REPORT miRNA reporter vector using the Sacl and HindIII sites. These constructs were validated by sequencing. Luciferase assay HEK-293 cells were seeded into a 24-well plate. After cultured overnight, cells were co-transfected with the wild-type and mutated p70S6K1 39-UTR reporter plasmid, and pRL-TK plasmids, or transfected with pre-miR-128 and miR-scrambled control precursors (miR-SCR). Luciferase assays were performed 48 h after transfection using the Dual Luciferase Reporter Assay System (Promega, WI, USA). MiRNA precursor transfection Pre-miR-128 and miR-SCR precursors were purchased from Applied Biosystems (Carlsbad, CA). Cells at 50–70% confluence were transfected using lipofectamine reagent (Invitrogen, CA, USA). Transfection complexes were prepared according to the manufacturer’s instructions. The final concentration of miR-128 or miR-SCR precursor for the transfection was 40 nmol/L. Immunoblotting After specific treatments as indicated, cells were washed with ice- cold PBS buffer, scraped from the dishes, and centrifuged. The supernatant was collected as the total cellular protein extracts and stored at 280uC. Cell lysates were prepared using RIPA buffer supplemented with protease inhibitors (100 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% deoxycholate acid, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2 mM DTT, 2 mM leupeptin, 2 mM pepstatin). Tumor tissues from human and nude mice were grinded into powder in liquid nitrogen with RIPA buffer, and the total tissue PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e32709