The T-NHL cell lines Jurkat and Hut-78 were used in these experiments. Jurkat and Hut-78 were maintained in the Key Clinical Laboratory of Henan Province. Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS (fetal bovine serum), 50 U/ml penicillin and 50 U/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C in a humidified atmosphere containing 5% CO₂.

From 2008 to 2011, 129 patients who were diagnosed with T-NHL and received standard chemotherapy after lymph node biopsy at The First Affiliated Hospital of Zhengzhou University were included in the study after obtaining their oral and written informed consent. The biopsy specimen of each patient was prepared by the Department of Clinical Pathology for paraffin-embedded tumor tissue sections. Information on histopathological diagnosis was extracted from medical records and reviewed by a specialist in gynecologic tumor pathology. The control group consisted of 17 samples of lymph node that were obtained from standard operations. This study was reviewed and approved by the ethics committee of the medical faculty at the First Affiliated Hospital of Zhengzhou University.

Antibodies against AEG-1, survivin, Bcl-2 and Bax were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-actin antibody and secondary goat anti-rabbit and goat anti-mouse alkaline phosphatase antibodies were also purchased from Santa Cruz Biotechnology. All antibodies were used for western blot analysis at a dilution of 1:2,000 and 1:5,000, respectively. RPMI-1640, FBS, Lipofectamine™ 2000 and TRIzol reagent were purchased from Invitrogen Co. (Carlsbad, CA, USA). MTT and DMSO were obtained from Sigma.

Standard immunoperoxidase procedures were used to visualize AEG-1 expression. Briefly, paraffin sections were deparaffinized in xylene, followed by a graded series of alcohols (100, 95 and 75%) and re-hydrated in water followed by Tris-buffered saline. Following antigen retrieval, slides were incubated with 3% H₂O₂ to inhibit endogenous peroxidase. Slides were then blocked with 5% normal serum and incubated with anti-AEG-1 antibody. After washing, the tissue sections were treated with biotinylated anti-rabbit secondary antibody (Zymed Laboratories Inc., South San Francisco, CA, USA), followed by further incubation with streptavidin-horseradish peroxidase complex (Zymed). Tissue sections were then immersed in 3,3′-diaminobenzidine and counterstained with 10% Mayer's hematoxylin, dehydrated and mounted.

AEG-1 expression in paraffin-embedded sections was viewed and scored separately by two independent pathologists, who were blinded to the histopathological features and patient data, and AEG-1 expression was determined by combining the proportion of positively stained tumor cells and the intensity of staining (15).

To quantify cell proliferation, Jurkat and Hut-78 cells and stably transfected cells (Jurkat-RNAi and Hut-78-RNAi) were seeded in a 96-well plate at a concentration of 2.5×10⁴/ml (100 μl/well). Six parallel wells were assigned to each group. Then, 20 μl/well of 5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added at 1, 2, 3, 4, 5 and 6 days after seeding and were then incubated for another 4 h. The supernatant was removed and the product converted from MTT was dissolved by adding 150 μl/well dimethylsulfoxide (DMSO). The plate was gently shaken for 15 min at room temperature and an enzyme-linked immunosorbent assay reader was used to measure the absorbance of each well at 570 nm. The growth curve was drawn according to the cell growth rate.

Total-RNA from cultured cells was extracted using the TRIzol reagent according to the manufacturer's instructions. The cDNA synthesis was carried out according to the protocol of the Takara Reverse Transcription System for real-time PCR [Takara Biotechnology (Dalian) Co., Ltd., China] with a starting amount of 2 μg RNA and reverse transcription performed with random primers. Real-time PCR primers were designed according to www.ncbi.nlm.nih.gov. Primer sequences are: AEG-1 forward, 5′-CTCGGGCTGCTGCTGCTGTT-3′, and reverse, 5′-CAGC AAGGCCAGGTCGTCGG-3′; Bax forward, 5′-GGCCGG GTTGTCGCCCTTTT-3′, and reverse, 5′-CCGCTCCCGGA GGAAGTCCA-3′; Bcl-2 forward, 5′-GGACGGGGTGA ACTGGGGGA-3′, and reverse, 5′-GGTGTGCAGGTGCCGG TTCA-3′; survivin forward, 5′-CGCTTTGCCACGGTGGT GGA-3′, and reverse, 5′-CAGGGGCGACATCTCCC GGT-3′; β-actin forward, 5′-CGACAACGGCTCCGGCATGT-3′, and reverse, 5′-TGGGCCTCGTCGCCCACATA-3′. Real-time PCR analysis was carried out on a LightCycler real-time PCR instrument using SYBR Green I kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. Each reaction was performed in triplicate. Data were analyzed with the Sequence detector software (v1.9, Applied Biosystems) and analyzed using the 2^−ΔΔCT method as previously described (16).

Total proteins were extracted by lysing cells in buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 25 mg/ml leupeptin and 25 mg/ml aprotinin. The lysates were cleared by centrifugation and the supernatants were collected. Proteins were extracted using the protein extraction kit according to the manufacturer's instructions. Protein concentration was determined using protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on SDS-PAGE, transferred to PVDF membranes, incubated with antibodies against AEG-1, survivin, Bcl-2 and Bax, β-actin, followed by incubation with the secondary antibodies. The membrane was then washed three times with TBST and visualized with diaminobenzidine. Quantification of the proteins was detected with the ECL system (Pierce Biotechnology Inc., Rockford, IL, USA). Each value represents the mean of triple experiments, and is presented as the relative density of protein bands normalized to β-actin.

The plasmid constructs carrying siRNA (pSUPER-EGFP-AEG-1) against AEG-1 were designed and constructed as previously described (17). The following siRNA sequences were cloned into the pSUPER-EGFP plasmid: forward, 5′-GCTGATCCCAACTCTGATTTTCAAGAGAAATCAGAGTTGGGATCAGC-3′, and reverse, 5′-CGACTAGGGTTGAGACTAAAAGTTCTCTTTAGTCTCAACCCTAGTCG-3′. The recombinant plasmids were transformed into competence bacillus coli and cultured; they were then extracted, purified, identified, measured and stored at −20°C. The T-NHL cell line Jurkat and Hut-78 cells were transfected with pSUPER-EGFP-AEG-1 using Lipo-fectamine 2000 (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, Jurkat and Hut-78 cells were diluted and selected in the medium containing 500 μg/ml G418 for 4 weeks. Then, the positive clones were picked up and expanded to establish cell lines after maintaining to select for 4 weeks. The stable transfection cell clones Jurkat-RNAi and Hut-78-RNAi were verified for RT-PCR and western blot analysis.

Flow cytometric analysis of PI-stained cells was performed to demonstrate the cell cycle of the different cell groups. Each different group of cells was cultured in a 10 cm dish, respectively. After culturing for 48 h, the cells were harvested, washed and fixed in 70% ethanol for 30 min. After fixation, cells were resuspended in 1 ml propidium iodide (PI) staining buffer (0.1% Triton X-100, 100 mg ml-1 RNase A, 500 mg ml-1 PI in PBS) at 37°C for 30 min and the proportion of cells in a particular phase of the cell cycle was determined by flow cytometry.

Annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, USA) was used to detect early apoptosis. In brief, after culturing for 48 h, each group of cells was harvested, washed twice with pre-chilled PBS and resuspended in binding buffer (HEPES-NaOH 10 mM pH 7.4, 144 mM NaCl and 25 mM CaCl₂) at a concentration of 1×10⁶ cells/ml. One hundred microliters of this solution (1×10⁵ cells) was mixed with 5 μl of Annexin V-FITC and 5 μl of PI (BD Biosciences) according to the manufacturer's instructions. The mixed solution was gently vortexed and incubated in the dark at room temperature (25°C) for 15 min. Four hundred microliters of 1X dilution buffer were added to each tube and cell apoptosis analysis was performed by flow cytometry (BD FACSCalibur) within 1 h. At least 10,000 events were recorded and represented as dot plots. Cells that stained positive for Annexin V-FITC and negative for PI were counted as apoptotic cells.

The activities of MMP-2 and MMP-9 tumor cells were assayed by gelatin zymography. Briefly, each group of cells was incubated with serum-free medium for 24 h. The conditioned medium was then harvested and concentrated by ultra-filtration centrifugation. The sample (20 μg) was mixed with loading buffer and subjected to gelatin zymogram gel (10% SDS-polyacrylamide gel, 0.1% gelatin). Electrophoresis was performed at 100 V for 3 h at 4°C. Following electrophoresis, gels were washed with washing buffer (2.5% Triton X-100 in ddH₂O) at room temperature to remove SDS, followed by incubation at 37°C in reaction buffer (40 mM Tris-HCl, pH 8.0, 10 mM CaCl₂, 0.02% NaN₃). After 16 h, the gels were stained with Coomassie blue R-250 (0.125% Coomassie blue R-250, 50% methanol, 10% acetic acid) for 3 h and destained with destaining solution (20% methanol, 10% acetic acid, 70% ddH₂O) until the clear bands were visualized. MMP-2 and MMP-9 (gelatinase) activity was visible as clear bands against the dark blue background, indicating proteolysis of the substrate protein, gelatin.

Each experiment was performed at least twice; results are expressed as mean ± SEM using Excel software (Microsoft, Redmond, WA, USA) for calculation. The SPSS13.0 software package (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. Statistical analysis was performed by ANOVA. Student's t-test was also performed and the results were considered statistically significant at P<0.05.

To examine whether the AEG-1 protein is upregulated in T-NHL tissues, 129 paraffin-embedded, archived T-NHL tissues were examined by immunohistochemical staining with an antibody against human AEG-1. As shown in Fig. 1, the AEG-1 protein was found to be upregulated in T-NHL compared with normal lymph node tissues. High levels of AEG-1 expression were present in areas containing tumor cells of the T-NHL. By contrast, AEG-1 was barely detectable in the normal lymph node tissues (P<0.01, Fig. 1).

Previous studies have shown that the expression of AEG-1 is associated with T-NHL growth. To further investigate the biological role of AEG-1 expression in T-NHL progression, the T-NHL cell lines Jurkat and Hut-78 were established to stably low-expressed AEG-1. Western blotting and RT-PCR assay results indicated that AEG-1-infected Jurkat-RNAi and Hut-78-RNAi cells have low AEG-1 mRNA and protein expression (P<0.01, Fig. 2A and B). The effect of AEG-1-siRNA on T-NHL cell proliferation was determined by MTT assay. As shown in Fig. 2, cell proliferation was remarkably inhibited in the Jurkat-RNAi and Hut-78-RNAi groups, when compared with the Jurkat and Hut-78 groups (P<0.05, Fig. 2C).

To analyze the mechanisms by which AEG-1-siRNA inhibits cell proliferation, the cell cycle of Jurkat and Hut-78 cells as well as transfected Jurkat-RNAi and Hut-78-RNAi cells were analyzed by flow cytometry. As shown in Fig. 3, the percentage of Jurkat and Hut-78 cells at the G0/G1 phase was increased from 54.95 and 48.52 to 69.52 and 67.19%; the S-phase cells were decreased from 32.63 and 36.4 to 21.36 and 24.44% (P<0.05, Fig. 3). These data show that silencing of AEG-1 can arrest cells at the G0/G1 phase.

To further evaluate whether knockdown of AEG-1 induces Jurkat and Hut-78 cell apoptosis, at 48 h after culture, the cells were harvested and analyzed by flow cytometry. As shown in Fig. 4, the apoptosis rates of the Jurkat-RNAi and Hut-78-RNAi groups were 13.2 and 11.51%, respectively, and were >1.64% in the Jurkat and 1.51% in the Hut-78 group (P<0.01, Fig. 4).

Since deregulation of the AEG-1 expression appears tightly linked to the apoptosis of Jurkat and Hut-78 cells, we further investigated whether survivin, Bcl-2 and Bax could be regulated by AEG-1. Western blot analysis revealed that ectopic expression of AEG-1 affects the expression of survivin, Bcl-2 and Bax, whereas the expression of Bax is upregulated and survivin and Bcl-2 is downregulated in Jurkat-RNAi and Hut-78-RNAi cells compared with control cells (P<0.01, Fig. 5A). Furthermore, real-time PCR results indicated that the modulation of survivin, Bcl-2 and Bax by AEG-1 were regulated at the transcriptional level (P<0.01, Fig. 5B). These data demonstrate that knockdown of AEG-1 may affect survivin, Bcl-2 and Bax expression, which all play an important role in tumor progression.

Since MMP-2 and MMP-9 play a critical role in tumor cell invasion, we examined the effects of AEG-1 silencing on the enzyme activities of MMP-2 and MMP-9 using gelatin zymography. Each group of cells was incubated in serum-free medium. After culturing for 24 h, conditioned media in each group were collected and the expression levels of interest were detected using zymography. The gelatinolytic activities of both MMP-2 and MMP-9 were found to be markedly reduced in the Jurkat-RNAi and Hut-78-RNAi groups compared with the control groups (P<0.01, Fig. 6). There was a significant difference between the groups (P<0.05).