Acta Biochim Biophys Sin, 2015, 47(6), 414–421 doi: 10.1093/abbs/gmv026 Advance Access Publication Date: 23 April 2015 Original Article

Original Article

Pituitary tumor transforming gene: a novel therapeutic target for glioma treatment Lishan Cui1,2, Songbai Xu1, Zhengmao Song2, Gang Zhao1,*, Xiaoqian Liu3,*, and Yuwen Song3 1

Department of Neurosurgery, The First Affiliated Hospital of Jilin University, Changchun 130021, China, 2Department of Neurosurgery, The Fifth Hospital of Xiamen, Xiamen 361101, China, and 3Department of Neurosurgery, The Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China *Correspondence address. Tel/Fax: +86-431-88785709; E-mail: [email protected] (G.Z.)/[email protected] (X.L.) Received 2 December 2014; Accepted 2 March 2015

Abstract Glioma which has strong proliferation and angiogenesis ability is the most common and malignant primary tumor in central nervous system. Pituitary tumor transforming gene (PTTG) is found in pituitary tumor, and plays important role in cell proliferation, cell cycle, cell apoptosis, and angiogenesis. However, the role of PTTG in glioma is still incompletely investigated. Here, we explored the correlation between PTTG and glioma grade, as well as micro-vessel density (MVD). In addition, siRNA was used to silence PTTG expression in glioma cell lines including U87MG, U251, and SHG44. Cell proliferation, apoptosis, invasion, and angiogenesis were studied both in vitro and in vivo. Our results demonstrated that PTTG expression was significantly up-regulated in glioma, and had positive correlation with glioma grade and MVD. Silencing of PTTG inhibited glioma cell proliferation, migration/invasion, and angiogenesis, induced cell apoptosis, suppressed cell invasion, and arrested cell cycle at G0/G1 stage. Silencing of PTTG could also inhibit tumor growth, invasion, and angiogenesis in vivo. Our data indicated that PTTG might be a potential target for glioma treatment. Key words: glioma, pituitary tumor transforming gene (PTTG), invasion, angiogenesis

Introduction Glioma is one of the most common and aggressive primary tumors in the brain. It shows unique biological features including special network of neo-plastic blood vessels, invasion, and metastasis. The current standard treatment schemes for high-grade glioma include gross total resection, radiotherapy, and concomitant and/or sequential chemotherapy. However, these therapy methods are not curative. With the development of molecular biology, the molecular genetic markers and chemotherapy resistance mechanisms of glioma have been extensively investigated. Since Folkman [1] proposed that tumor growth and metastasis depend on angiogenesis, the inhibition of angiogenesis has been extensively studied, which has been proved to be the most effective method for non-operative treatment of glioma.

Pituitary tumor transforming gene (PTTG) which is underexpressed in normal pituitary but over-expressed in pituitary tumor was discovered in 1997 [2]. PTTG family has three members [3,4], but only PTTG1 is the most crucial and extensively studied one. PTTG locates at 5q33, its cDNA is 787 bp, which was first isolated from cDNA library in 1998 [5], and the molecular weight of PTTG protein is 22–23 kDa [4]. Human PTTG protein was first isolated from fetus liver [6]. It is expressed in special tissues/organs such as testis, thymus gland, and spleen, which had high proliferation activities. But in differentiated mature tissue, PTTG expression could hardly be detected [5,7–9]. According to the previous studies, PTTG plays an important role in gene modulation, angiogenesis, mitoses, cell cycle control, cell transformation, DNA repair, and cell apoptosis [10–12]. High level of PTTG protein is detected in most malignances,

© The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 414

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such as esophagus cancer, breast cancer, and cervical cancer, suggesting that PTTG is a potential oncogene involved in tumor development, angiogenesis, and lymphatic metastasis [13]. However, the role of PTTG in glioma is still unclear. In this study, we tried to reveal the relationship between PTTG expression and glioma grade, as well as the association between PTTG expression and micro-vessel density (MVD). PTTG expression was silenced in glioma cell lines with specific siRNA to investigate its biological functions, which may provide a potential target for glioma treatment.

expression level. Cells transfected with empty pGPU6/Neo vector served as NC.

Materials and Methods

MTT assay Glioma cells were seeded in 96-well plates (6000 cells per well) and transfected as described earlier. After transfection, cells were incubated for 24, 48, or 72 h, and cell viability was analyzed by methyl thiazolyl tetrazolium (MTT) assay. Briefly, cells were incubated with MTT solution (1 mg/ml in high glucose DMEM) at 37°C for 4 h. Then, the medium was decanted and 150 µl DMSO was added into each well. Absorbance was measured at 490 nm using a Microplate Reader (Bio-Rad, Hercules, USA). Assays were performed in triplicate.

Tissue collection Glioma samples were obtained from 72 patients (43 male, 29 female, aged from 5 to 71 years) who had surgical resection of glioma between 2008 and 2010. Normal brain tissues were obtained from 20 patients (12 male, 8 female, aged from 20 to 45 years) who underwent decompressive surgical procedures for severe head injury within the same time period. The protocol of this study was approved by the Ethics Committee of our hospital. Gliomas were of different histological grades: 12 pilocytic astrocytomas (WHO I), 20 low-grade astrocytomas (WHO II), 20 anaplastic astrocytomas (WHO III), and 20 glioblastomas (WHO IV).

Western blot analysis Total protein was extracted and 50 μg total protein was loaded each lane. Then, electrophoresis was performed using 6%–10% SDS– PAGE. Proteins on the gel were transferred onto polyvinylidene difluoride membrane (Millipore, Bedford, USA), followed by blocking with 5% skimmed milk dissolved in TBS containing 1% Tween-20 (TBST) for 1 h at room temperature. The membrane was incubated with primary antibody at 4°C overnight and incubated in alkaline phosphatase-conjugated secondary antibody for 1 h at room temperature after three times wash with TBST. Finally, specific proteins were detected with ECL plus kit (ZhongShan Co., Ltd.).

Immunohistochemistry All tumor tissues were fixed with formalin and then embedded with paraffin, and sliced into 4-μm sections. After deparaffinization and hydration, the slides were treated with 0.3% peroxide for 15 min, and blocked with 1.5% blocking serum (Invitrogen, Carlsbad, USA) for 2 h at room temperature. A rabbit anti-human PTTG polyclonal antibody (Abgent, San Diego, USA) was used for overnight incubation. After being washed with phosphate-buffered saline (PBS) three times and incubated with horseradish peroxidase-conjugated goat antirabbit IgG secondary antibody at 37°C for 30 min, the slides were treated with diaminobenzidine (ZhongShan Co., Ltd., Beijing, China). Finally, the slides were lightly counterstained with hematoxylin, dehydrated, and mounted. The mean percentage of PTTG-positive cells of each sample was determined by counting at least five random visual fields with a microscope at 400× magnification. After detection of PTTG expression, all tumor tissues were subject to MVD examination by CD31 (Abgent) staining. The mean percentage of MVD of each sample was determined by counting at least five random visual fields with a microscope at 400× magnification. For negative controls (NCs), primary antibodies were either omitted or replaced by non-specific mouse IgG.

Cell culture and siRNA transfection Glioma cell lines U251, U87MG, and SHG44 were obtained from Shanghai Cell Collection (Shanghai Institutes for Biological Sciences, Shanghai, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin. siRNA sequences targeting PTTG were designed and synthesized by Shanghai Ruigene Bio-Pharm (Shanghai, China). Then, the siRNA was ligated to pGPU6/Neo vector. The cells were transfected with plasmid containing siRNA with Lipofectamine 2000 (Invitrogen) after 1-day culture in DMEM without FBS and antibiotics. The culture medium was changed to complete medium 8 h after transfection. The transfection efficiency was examined by immunofluorescence, PTTG protein expression, and mRNA

Real-time polymerase chain reaction Cells or tissues were lysed with Trizol reagent (Invitrogen) and total mRNA was extracted. The mRNA was reverse transcribed into cDNA with a reverse-transcription kit (Promega, Madison, USA). For polymerase chain reaction (PCR) analysis, cDNA from triplicate dishes was diluted to a final concentration of 10 ng/μl. Quantitative real-time PCR was performed with a Universal Master Mix (Chembase, Beijing, China). cDNA (50 ng) was used to determine the relative amounts of mRNA by real-time PCR (MAX3000 Sequence-Detection System; Chambase) using specific primers and probes. The reaction was conducted for 40 cycles. β-Actin was used as a reference.

Transwell assay Appropriate Matrigel (BD Biosciences, Bedford, USA) was added to the upper chamber of the transwell apparatus with 8 μm pore size membrane (Costar, Cambridge, USA). After the Matrigel solidified at 37°C, serum-free DMEM containing 1 × 105 siRNA-transfected and control U87MG cells in 100 μl was added into the upper chamber. The lower chamber was loaded with 500 μl medium with 10% FBS. After incubation at 37°C for 24 h, membranes coated with Matrigel were wiped with a cotton swab and fixed with 100% methanol for 10 min. The membranes with cells were soaked in 0.1% crystal violet for 10 min and then washed with distilled water. The number of cells attached to the lower surface of the polycarbonate filter was counted at 400× magnification under a light microscope. Results were expressed as the mean of three experiments.

Vasculogenic mimicry assay in vitro Immediately before use, 24-well plates were coated with highconcentration of Matrigel (200 μl/well) and incubated at 37°C for 40 min, until the Matrigel was solid. Glioma cells were transfected and pre-incubated in medium without serum overnight. Cells were lifted with 0.05% trypsin that was neutralized with DMEM with 10% FBS. Cells were then spun down, re-suspended and seeded into

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Matrigel-coated wells at the density of 3 × 104 cells/well. Photomicrographs were taken after 16 h of incubation from each well and the number of tubes (complete circular structures) was counted. The mean from three readings of each well was used as the final reading from that well. human umbilical vein endothelial cell was used as a reference.

glioma, III, and IV glioma were 20%, 67%, 80%, 85%, and 100%, respectively. The number of PTTG-positive cells was increased with increasing histological grade of tumor (Fig. 1A and Table 1). MVD in higher grade glioma was much higher than that in lower grade glioma (Table 2; P < 0.05). However, the MVD was associated with PTTG expression, as there were more microvessels in PTTG strong positive tissue than that in the weak positive tissue (Fig. 1B and Table 3). These results demonstrated that PTTG is associated with glioma grade and MVD.

Cell apoptosis and cell cycle assay After designated treatment, U87MG cells were washed, harvested, and counted. About 1 × 105 cells were re-suspended in 100 μl binding buffer and incubated in the dark for 15 min at room temperature, then 10 μl of Annexin V and 5 μl of propidium iodide (PI) were added according to the manufacturer’s instruction (Biosea, Beijing, China). The apoptosis rate (%) was determined with a cytometer (Epics Altra II; Beckman Coulter, Danvers, USA). Experiment was repeated three times. After designated treatment, U87MG cells were harvested, washed with ice-cold PBS, and fixed with 70% ethanol at 4°C overnight. The ethanol was removed by centrifugation and ∼1 × 106 cells were resuspended in PBS containing PI (50 μg/ml; Sigma, St Louis, USA) and RNase A (50 μg/ml; Sigma), and incubated for 30 min in the dark before analyzed by flow cytometry (FACScalibur; Becton Dickinson, Franklin Lakes, USA). The percentage of cells at G0/G1, S, or G2/M phase was thereby calculated. DMSO-treated cells were used as controls. Experiment was repeated three times.

Animal experiment In animal experiments, all surgical procedures and care administered to the animals were in accordance with institutional guidelines. A total of 36 male nude BALB/c mice (6–8 weeks old) were obtained from the Animal Research Center, Bethune Medical College of Jilin University (Changchun, China). Tumors were established by subcutaneous injection of 5 × 106 U251 cells into the flanks of mice. Twenty-one days after injection of tumor cells, 5 μg PTTG-siRNA plasmid or NC plasmid diluted in 100 μl PBS was injected into the center of the tumor daily for 28 days. Tumor size was measured twice a week and tumor volumes were estimated according to the formula: π/6 × A 2 × B, where A is the short axis and B is the long axis. The mice were closely monitored for 28 days before euthanized. Each tumor tissue was split into two pieces: one was fixed in 10% buffered formalin for immunohistochemistry, and the other was stored at −80°C for western blot analysis.

Periodic acid–Schiff staining In periodic acid–Schiff (PAS) staining, the samples were stained with PAS followed by counterstaining with hematoxylin. Finally, the slides were dehydrated and mounted. The mean percentage of PTTG-positive cells and VM structures of each sample was determined by counting at least five random visual fields with a microscope at 400× magnification.

Statistical analysis Data were shown as the mean ± standard deviation (SD) from at least three independent experiments. Statistical analyses were performed by SPSS software 13.0 (SPSS, Chicago, USA). A P-value of