Anti-TrioBP antibodies were purchased from Novus Biologicals, LLC (Littleton, CO, USA) and anti-actin antibodies were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibodies were purchased from Komabiotech (Seoul, Korea). The siRNA against human TrioBP were synthesized by Intergrated DNA technologies. The siRNA sequences for TrioBP were the following; 5′-TCCCAGCAGAACCATCCAACAAGAGAA-3′.

The glioblastoma cells (U87, U25, and U343-MG) were maintained in medium (RPMI) supplemented with 10% FBS, 25 mM HEPES (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% Antibiotics-Antimycotics (Thermo Fisher Scientific, Inc.). U87 and U251-MG cells were transiently transfected with 30 nM control siRNA or TrioBP siRNA by using Neon Transfection System (Thermo Fisher Scientific, Inc.).

The study was approved by the Hospital Institutional Review Board (approval number CNUH 2013-11-006) according to the Declaration of Helsinki at Chungnam National University Hospital (Daejeon, Korea), and written informed consent was obtained from each patient before surgery. Normal brain tissue samples were obtained from cadavers alternatively, from autopsy of surrounding normal brain of glioblastoma patient who underwent surgery.

The immunoblot analysis was performed as the described previously (17,18). Briefly, cells were placed on ice and extracted with lysis buffer containing 50 mM Tris-HCl, pH 7.5, 1% v/v Nonidet P-40, 120 mM NaCl, 25 mM sodium fluoride, 40 mM β-glycerol phosphate, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 2 µM microcystin-LR. Lysates were centrifuged for 15 min at 12,000 × g. The cell extracts were resolved by 10–15% SDS-PAGE, and transferred to Immobilon-P membranes (EMD Millipore, Billerica, MA, USA). The filters were blocked for 1 h in 1X Tri-buffered saline buffer (TBS; 140 mM NaCl, 2.7 mM KCl, 250 mM Tris-HCl, pH 7.4), containing 5% skimmed milk and 0.2% Tween-20, followed by an overnight incubation with the anti-TrioBP and anti-actin antibodies diluted 1,000-fold at 4°C. The secondary antibody was horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Koma Biotech, Seoul, Korea), diluted 5,000-fold in the blocking buffer. The detection of protein expression was visualized by enhanced chemiluminescence, according to the manufacturer's instructions (Thermo Fisher Scientific, Inc.).

Total RNA was extracted from frozen tissue samples or from cells using the PureHelix RNA Extraction Solution (Nanohelix, South Korea). The cDNA was synthesized from total RNA with the SuperScript III First-Strand Synthesis System for RT-qPCR (Invitrogen; Thermo Fisher Scientific, Inc.). The RT-qPCR measurement of individual cDNAs was performed using SYBR-Green dye to measure duplex DNA formation with the StepOne Plus real-time PCR system (Invitrogen; Thermo Fisher Scientific, Inc.) and normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RNA. The following primers were used in the RT-qPCR (forward, reverse); human TrioBP: F-5′-TCCAAGGTCTCCCTTAGTACA/R-5′-GTGGGACTGGACTTGCTA; human GAPDH: F-5′-TCGACAGTCAGCCGCATCTTCTTT/R-5′-TACGACCAAATCCGTTGACTCCGA.

Total RNA of U87-MG, U251-MG and normal brain was extracted using TRIzol reagent (Invitrogen) following the manufacturer's procedures. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Roughly 10 µg of total RNA was subjected to isolate Poly (A) mRNA with poly-T oligo attached magnetic beads (Invitrogen; Thermo Fisher Scientific, Inc.). Following purification, the mRNA is fragmented into small pieces using divalent cations under raised temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, Inc., San Diego, CA, USA). The average insert size for the paired-end libraries was 300 bp (± 50 bp). Next we performed the paired-end sequencing on an Illumina Hiseq 2000 system at Macrogen (Seoul, Korea) following the vendor's recommended protocol. For each sample, sequenced reads were aligned to the UCSC human reference genome (19) using the Tophat package (20), which initially removes a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome. FPKM (fragments per kilobase of exon per million fragments mapped) were calculated to compare the expression level of TrioBP mRNA variants in each sample.

U251-MG cells were grown on glass coverslips until they were 50–70% confluent. After 24 h, the cells were fixed in 4% paraformaldehyde at room temperature for 10 min and permeabilized in 0.2% Triton X-100 for 5 min at room temperature. Then cells were incubated in blocking buffer containing 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) in 1X TBS for 1 h at 37°C. The rabbit polyclonal anti-TrioBP was diluted 200-fold for primary antibody and incubated for overnight. The secondary antibody, FITC-conjugated anti-rabbit antibody (BD Biosciences, Franklin Lakes, NJ, USA) was used. After appropriate rinsing, cover slips were mounted with Vectashield (Vector Laboratories, Inc., Burlingame, CA, USA) and visualized using a Zeiss confocal microscope (Zeiss AG, Oberkochen, Germany).

The analysis of immunohistochemistry was performed as the described previously (21). A human cancer tissue array slide with paraffin sections was purchased from Bio Max (US Biomax Inc; Thermo Fisher Scientific, Inc.). Histostain-Plus kits (Zymed Laboratories Inc., San Francisco, CA, US) were used in accordance with the manufacturer's instructions for the immunohistochemistry of tissue array. Briefly, paraffin sections were deparaffinized with xylene and rehydrated in a graded series of ethanol. The slide was submerged in peroxidase quenching solution for 10 min. After it was washed twice with PBS for 5 min, it was added with 2 drops of Reagent A for blocking and incubated for 30 min. Following two washes with PBS, the primary antibody was applied at 4°C for overnight. Then biotinylated secondary antibody, Reagent B, was added after rinsing with PBS. It was incubated at room temperature for 1 h. It was rinsed with PBS and dropped with enzyme conjugated Reagent C. After it was washed with PBS, DAB chromogen, and a mixture of Reagent D1, D2, and D3, it was dropped, and signals were observed with a florescence microscope. Then the reaction was stopped with distilled water, and pictures were taken with a microscope.

Glioma data sets and corresponding clinical data were downloaded from the publicly available databases (213 cases from the TCGA GBM dataset (http://www.betastasis.com/glioma/tcga_gbm/)). Normal; n=11, Classical; n=54, Mesenchy-mal; n=58, Neural; n=33, Proneural; n=57.

Real-time assay for cell proliferation and migration were measured using an xCELLigence RTCA DP system (Roche Diagnostics, Indianapolis, IN, USA), which monitors cellular events in real-time without the incorporation of labels. Briefly, cells were placed into well of an E-plate 16 (for proliferation; U87-MG 2×10⁴, U251-MG 1.2×10⁴ cells) or Matrigel-coating (Matrigel: serum free media, 1:40) of the upper chamber of CIM-plate 16 (for migration; U87-MG 2×10⁴, U251-MG 1.2×10⁴ cells) and incubated for indicated times.

Data are expressed as the mean ± SD from at least three separate experiments performed triplicate. The differences between groups were analyzed using a Student's t-test and P<0.05 was considered significant, and P<0.01 was highly significant compared with corresponding control values. Statistical analyses were carried out using SPSS software ver. 13.0 (SPSS, Inc., Chicago, IL, USA). Other statistical analysis performed using analysis of variance (ANOVA) and Tukey's post hoc tests, using the GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Differences were considered significant if P<0.05, P<0.01, and P<0.001.

Immunohistochemical (IHC) analysis using a tissue array and anti-TrioBP antibody showed stronger signals in brain tumor tissues than in surrounding normal tissues (Fig. 1A). Total lysates of normal and cancerous tissues from GBM patients were subjected to immunoblotting using an anti-TrioBP antibody. TrioBP levels in GBM tissues were higher than those in the surrounding normal tissues (Fig. 1B), suggesting that TrioBP expression is upregulated in glioblastoma patients.

Based on the high TrioBP expression in samples from GBM patients (Fig. 1), TrioBP expression in glioblastoma cell lines was assessed by immunoblotting. TrioBP expression was significantly increased in U87-MG and U343-MG cells than in the other cell lines (Fig. 2A). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showed that the mRNA level of TrioBP was increased in U343-MG, and U87-MG cells compared with that in the controls (Fig. 2B).

As TrioBP expression was elevated in glioblastoma cells (Fig. 2A and B), the TrioBP mRNA levels in glioblastoma cell lines were determined by transcriptome profiling. Total RNA was prepared from U251-MG (low TrioBP expression) and U87-MG (high TrioBP expression) glioblastoma cells. Next, mRNA was split into small fragments to produce a cDNA library. The number of fragments per kilobase of exon per million fragments mapped (FPKM) was considered to be representative of the TrioBP mRNA level. FPKM values were greater in U87-MG cells (34.98) and U251-MG cells (27.58) than in cerebral cortex cells (11.87) (Fig. 3), demonstrating that TrioBP transcription is enhanced in glioblastoma cells.

To investigate the roles of TrioBP in cancer cells, U251-MG and U87-MG cells were transfected with TrioBP siRNA and their proliferation and migration were evaluated by Excelligence. TrioBP expression was significantly reduced (Fig. 4A). siRNA-mediated knockdown of TrioBP inhibited the proliferation of U87-MG cells (left) and U251-MG cells (right) (Fig. 4B). The reduction in proliferation of U87-MG cells (left) was greater than that of U251-MG cells (right). In addition, the migration of U87-MG and U251-MG cells was suppressed (Fig. 4C). Therefore, TrioBP may play important roles in cancer cells.

To investigate further its roles in GBM cells, the intracellular localization of TrioBP in U251-MG cells was evaluated. Interestingly, TrioBP was distributed in a punctate pattern, possibly associated with the cytoskeleton and cytosol (Fig. 5), indicating that localization to the cytosol is important for its function in glioblastoma cells.

A bioinformatic analysis was performed on the TCGA GBM cohort. Similar to the above results, TrioBP expression was increased in classical (n=54), mesenchymal (n=58), neural (n=33), and proneural (n=57) glioblastomas compared with the level in normal controls (n=11; one-way analysis of variance, P<0.0001; Fig. 6).