A total of 24 brain glioma tissue samples and their matched adjacent normal tissues from 24 patients obtained following surgical resection were collected from the Department of Neurosurgery, Yantai Yuhuangding Hospital Affiliated to Qingdao University Medical College (Yantai, China) between August 2010 and September 2012. Adjacent tissues were located 1 cm away from lesions. All specimens were obtained under sterile conditions during surgery, and immediately placed into Eppendorf tubes and frozen at −80°C. The present study was approved by the ethics committee of Shandong University (Jinan, China; approval no. 20130041). Written informed consent was obtained from all patients.

The human astrocyte HA cell line was purchased from ScienCell Research Laboratories (San Diego, CA, USA). The human glioma cell lines U251, U87, LN-18 and H4 were all purchased from the American Type Culture Collection (Manassas, VA, USA). All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with heat-inactivated 10% fetal calf serum (GE Healthcare Life Sciences), 2 mM L-glutamine and 100 U/ml penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 5% CO₂.

The lentivirus for PDCD4 overexpression (Lenti-PDCD4) and the control (Lenti-Empty) were commercially constructed by Genechem Co., Ltd. (Shanghai, China). The lentivirus was packaged in HEK-293T cells and collected from the supernatant following the manufacturer's protocol. Glioma cells were infected with lentiviral particles. Cell lines stably expressing PDCD4 were established using puromycin as the selection marker.

Small interfering RNA (siRNA) targeting HOTAIR was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Cells were transfected using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol.

RNA was isolated from the human glioma cell lines using RNAzol^® reagent (Vigorous Biotechnology Co., Ltd., Beijing, China), according to the manufacturer's protocol. The RNA was treated with DNase H (Beyotime Institute of Biotechnology, Haimen, China) to remove contaminating genomic DNA. cDNA was synthesized in a 25 µl reaction mixture consisting of 2 µg total RNA, 1 µl M-MLV Reverse Transcriptase 2 µl dNTPs, 5 µl 5X buffer, 1 µl random primers, 0.5 µl RNasin and diethylpyrocarbonate (all Promega Corporation, Madison, WI, USA). qPCR was performed using the ABI 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with reagents from the SYBR^® Green Real-Time PCR Master Mix (Toyobo Co., Ltd., Osaka, Japan) and the appropriate primers, which are presented in Table I. The PCR cycling conditions were as follows: 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. Relative mRNA expression levels were determined following normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the 2^−ΔΔCq method (17).

Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to determine cell proliferation rate according to the manufacturer's protocol at the indicated time points. Briefly, cells were seeded in 96-well plates at a density of 2,000 cells/well. Cell proliferation reagent (10 µl) was added to each well, and the cells were incubated for 2 h at 37°C. Cell numbers were estimated by measuring the optical density at 450 nm. The absorbance of cell-free wells containing medium was set as zero. Data was obtained from three separate experiments and three replications were performed each time.

Transwell chambers (8.0 µm pore size; Corning Incorporated, Corning, NY, USA) coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were used to measure the invasiveness of glioma cells. In brief, 5×10⁴ cells/well were seeded in the upper chamber in DMEM without serum, and the lower chamber contained DMEM supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) to stimulate cell invasion. Following 48 h of incubation at 37°C, cells that migrated to the bottom of the chamber insert were fixed with 3% paraformaldehyde, stained with 0.1% crystal violet, extracted with 33% acetic acid and finally detected quantitatively using a standard microplate reader (at 570 nm). Data was obtained from three separate experiments and three replications were performed each time.

Protein extracts (10 µg) prepared with radioimmunoprecipitation assay buffer were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes by electroblotting. Subsequent to blocking with 5% non-fat milk, the membranes were incubated overnight at 4°C with mouse anti-PDCD4 (#sc-376430) and mouse anti-GAPDH (#sc-25778) monoclonal antibodies (dilutions, 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Blots were then incubated with peroxidase-conjugated goat anti-mouse secondary antibodies (dilution, 1:1,000; #ZB2305 and #ZB2307; Beijing Zhongshan Jinqiao Biotechnology, Co., Ltd., Beijing, China) for 1 h at room temperature and developed using a SuperSignal™ West Pico Chemilumiscent substrate (Pierce Biotechnology, Inc., Rockford, IL, USA). Immunoblots were scanned using Image Lab™ software, version 1709690 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cells were fixed with 10% formaldehyde and sonicated to prepare the chromatin sample. Chromatin samples were immunoprecipitated with mouse anti-H3K27me3 monoclonal antibody (#ab6002; Abcam, Cambridge, MA, USA), rabbit anti-enhancer of zeste homolog 2 (EZH2) polyclonal antibody (#ab3748; Abcam) or rabbit immunoglobulin G (IgG; #ab6785; Abcam) at 4°C for 3 h. Following crosslink reversal, precipitated DNA was analyzed by PCR for fragments of the PDCD4 promoter using the following primers: Forward, 5′-GGGAGGAGGAATCGGACAG-3′; and reverse, 5′-TATGTTGGGAGGCGTGGC-3′ (141 bp). The PCR cycling conditions were as follows: 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. The data obtained were normalized to those of corresponding DNA precipitated by IgG.

All data are expressed as the mean ± standard deviation. Comparisons between two groups were performed using Student's t-test or among groups with one-way analysis of variance. Statistical analyses were conducted using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Human glioma tissue samples (n=24) were analyzed to detect the change in expression of PDCD4 in glioma. RT-qPCR demonstrated that PDCD4 was significantly downregulated in glioma tissues (by ~20%), as compared with adjacent normal tissues (P=0.034; Fig. 1A), which was supported by western blot analysis (Fig. 1B). In a few samples, no PDCD4 expression was detected.

This experiment was repeated with glioma cell lines (U251, LN-18, U87 and H4), and the human astrocyte HA cell line was used as the control. RT-qPCR and western blot analysis demonstrated that PDCD4 expression was suppressed in all glioma cell lines compared with the HA cells, and its expression was the lowest in the U251 cells (Fig. 1C and D).

Next, the potential functions of PDCD4 in glioma were investigated. A lentivirus was constructed, Lenti-PDCD4, containing the full length PDCD4 cDNA, and PDCD4 was overexpressed in U251 cells by infection with Lenti-PDCD4. Western blot analysis indicated that PDCD4 was successfully overexpressed compared with the Lenti-Empty construct (Fig. 2A). CCK-8 assay was employed to determine whether PDCD4 affects the proliferation of glioma cells. Glioma cells infected with Lenti-PDCD4 exhibited a significantly lower proliferation rate than the control at 48 and 60 h following transfection (P<0.05; Fig. 2B). In addition, Transwell migration assays were performed to verify invasive ability. The results demonstrated that PDCD4 overexpression resulted in a significant decrease in the invasion rate of U251 cells compared with the control (P<0.05; Fig. 2C).

Next, mechanisms underlying PDCD4 downregulation in glioma were investigated. Epigenetic modifications, particularly methylation at specific histone sites, are important in gene expression. ChIP assays demonstrated that the level of H3K27me3 at the PDCD4 promoter region increased significantly in the U251 cells, as compared wih the HA cells (P=0.019; Fig. 3A), thus favoring transcriptional silencing. Conversely, H3K4me3 exhibited little change between the HA and U251 cells (P>0.05; Fig. 3A). The levels of EZH2 and suppressor of zeste 12 (SUZ12), core components of the PRC2 complex, at the PDCD4 promoter region were significantly increased in the U251 cells, as compared with the HA cells (P<0.05; Fig. 3B). These results indicated that the PRC2 complex was able to downregulate PDCD4 expression by increasing the level of H3K27me3 at its promoter.

The expression of HOTAIR was measured in human glioma tissue samples. RT-qPCR demonstrated that HOTAIR expression was significantly elevated (by ~30-fold) in glioma tissues, as compared with normal tissues (P=0.039; Fig. 4A). A similar trend was observed in the glioma cell lines, in which HOTAIR RNA levels were significantly elevated compared with the HA cells (P<0.05; Fig. 4B). To investigate the function of HOTAIR, the gene was knocked down in U251 cells using siRNA, which significantly decreased its expression, as compared with the scramble RNA (P<0.05; Fig. 4C).

The present study investigated how the expression of PDCD4 was silenced in glioma cells. It was hypothesized that HOTAIR may induce the recruitment of PRC2 to the PDCD4 promoter. Western blot analysis indicated that PDCD4 expression was upregulated when HOTAIR was knocked down in glioma cells (Fig. 5A). Histone modifications at the PDCD4 promoter were measured by ChIP assays. The results demonstrated that when HOTAIR was knocked down in glioma cells, the H3K27me3 level at the PDCD4 promoter was significantly reduced compared with the control (P=0.031; Fig. 5B). Furthermore, the recruitment of EZH2 and SUZ12 to the PDCD4 promoter was measured, and the results indicated that the level of PRC2 components at the PDCD4 promoter was decreased in glioma cells following HOTAIR-knockdown compared with the control (Fig. 5C). These results suggest that the upregulation of HOTAIR results in PDCD4 silencing in glioma cells.