Unless stated otherwise, all substances were purchased from Gibco (Grand Island, NY, USA). RPMI-1640 medium was purchased from Sigma (St. Louis, MO, USA). Rabbit anti-LMP-1, eIF4E, NF-κB and IκB monoclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-actin antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). HRP-conjugated goat anti mouse and anti-rabbit antibodies were from Abcam (Cambridge, MA, USA).

The SNK-6 cell line was purchased from ScienCell (San Diego, CA, USA). Cells were cultured in RPMI-1640 media supplemented with 10% heat-inactivated human plasma, 700 U/ml of recombinant interleukin-2 (IL-2; Novartis, Surrey, UK), 50 U/ml penicillin and 50 µg/ml streptomycin. PBMCs from healthy volunteers were isolated by centrifugation using Ficoll-Hypaque (Amersham Pharmacia Biotech, Buckinghamshire, UK). Both cell lines were incubated at 37°C in an atmosphere containing 5% CO₂. Cells used for the experiments were all in the logarithmic phase.

The shRNA-LMP1/eIF4E and its control shRNA-NC plasmids were designed and synthesized by Shanghai GeneChem Chemical Technology Co., Ltd. (Shanghai, China). The stable LMP1/eIF4E knocked down cell line was established via lentiviral vector transfection. SNK-6 cells in the logarithmic growth phase were cultured with lentiviral vector solution for 6 h, and supplemented with lentiviral vector for another 6 h. After 48 h, cells were selected with hygromycin B until positive cells were identified.

The pcDNA3.1-LMP1 plasmid was constructed by inserting the cDNA fragment retro transcribed from the full-length cDNA of LMP1 (16). Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) was used for the transfection of LMP1 plasmid (pcDNA3.1-LMP-1) and empty plasmid (pcDNA3.1) according to the manufacturer's protocol. Stable expression clones were selected using G418 (neomycin sulfate, 800 µg/ml). The cell culture medium was replaced with fresh G418-containing medium every 2–3 days until resistant colonies were identified. The cells were collected for further analysis.

Cell viability was monitored via the 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) colorimetric assay. Briefly, after adding 20 µl of MTT (5 mg/ml) to each well, and a 4-h incubation at 37°C, MTT crystals were dissolved with dimethyl sulfoxide (DMSO) without discarding the cell supernatants and the absorbance (490 nm) was measured. All experiments were repeated at least three times with 96-wells per experiment.

For cell cycle analysis, SNK-6 cells were cultured in 0.01 mmol/l doxorubicin, or not. After 24 h, the cells were stained in 50 mg/ml propidium iodide, 0.05% Triton X-100, 0.1 mg/ml RNase A, and 1X PBS at 37°C for 30 min in the dark. The stained cells were suspended in 500 ml PBS for flow cytometric analysis.

Cell apoptosis was analyzed using an Annexin V-PI apoptosis detection kit (Abcam, Cambridge, UK). Briefly, transfected cells were washed with PBS and resuspended in 500 µl of binding buffer containing Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). After incubation on ice for 10 min, cells were analyzed on a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). The relative number of apoptotic cells was calculated.

The BioCoat Matrigel Invasion Chamber was purchased from BD Biosciences (Bedford, MA, USA). The membrane has a pore size of 8 mm and is coated with Matrigel matrix. According to the manufacturer's instructions, 1×10⁶ SNK-6 cells in 0.5-ml culture medium were implanted into the upper chamber. The bottom well contained 0.6 ml of Dulbecco's modified Eagle's medium with 10% fetal bovine serum. After being cultured for 48 h, the cells in the upper and lower chambers were stained with 0.4% trypan blue and counted with a hemocytometer. After air-drying, the membrane was stained with crystal violet. The number of migrating cells was then counted under a microscope.

An invasion assay was carried out similarly to the cell migration assay, except that 0.1 ml of Matrigel (50 mg/ml; BD Biosciences) was added to the membrane surface of the chamber 6 h before the cells were seeded.

Cells were harvested after transfection or 48 h later. Total RNA of the cells was extracted using TRIzol reagent (Invitrogen). The cDNA was synthesized from 5 µg of the total RNA using M-MLV reverse transcriptase (Clontech Laboratories, Palo Alto, CA, USA). The obtained cDNA was then used as a template for qRT-PCR analysis. First strand cDNA was synthesized from 2 µg of total RNA using the SuperScript II reverse transcriptase (Invitrogen) with 200 ng of random hexamers. The qRT-PCR cycling conditions were as follows: 95°C for 2 min for initial denaturation; 94°C for 15 sec, 58°C for 15 sec, and 72°C for 20 sec; 2 sec for plate reading for 40 cycles; and melt curve from 65 to 95°C. The primers used were as follows: LMP-1, 5′-GGT ACC TAC ATA AGC CTC TCA CAC TG-3′ (forward primer) and 5′-TCT AGA GAA GGT AAG AGT GCC ATC-3′ (reverse primer); eIF4E, 5′-GGG CCC ATG GCG ACT GTC GAA CCG GA-3′ (forward primer) and 5′-CTC GAG TTA GTG GTG GAG CCG CTC TTA-3′ (reverse primer); β-actin, 5′-CTG GGA CGA CAT GGA GAA AA-3′ (forward primer) and 5′-AAG GAA GGC TGG AAG AGT GC-3′ (reverse primer).

After transfection for 48 h, cells were harvested after being washed with cold PBS (Invitrogen). Total protein was extracted using the RIPA lysis buffer system (Santa Cruz Biotechnology, Dallas, TX, USA). A micro-BCA protein assay kit (Pierce, Rockford, IL, USA) was used for protein concentration analysis. Total protein (30 µg/lane) was resolved on a 12% SDS/PAGE under denatured reducing conditions and transferred onto a nitrocellulose membrane (Amersham Pharmacia, Freiburg, Germany). After 1-h incubation in blocking solution (5% non-fat milk), the membrane was probed overnight at 4°C with primary antibodies [LMP1 (1:500), eIF4E (1:300), NF-κB (1:400), IκB (1:400), β-actin (1:500)]. The membranes were washed five times then incubated with HRP-conjugated secondary antibodies (1:2,000) for 1 h at room temperature. Chemiluminescent detection was performed using an ECL kit (Pierce Chemical, Rockford, IL, USA). The gray value of the bands was analyzed using Image J2x software.

Each experiment was repeated at least three times. The results are presented as the mean ± standard deviation (SD). Statistical analyses were performed using an independent samples Student's t-test for direct two-group comparisons and the Tukey-Kramer test after a significant one-way analysis of variance (ANOVA) F-test for multiple-group comparisons. A difference was considered statistically significant at P<0.05.

It has been demonstrated that LMP1 is associated with the development of malignancies (17,18). To investigate its function in NKTL progression, its levels were measured. RT-PCR analysis showed that the mRNA levels of LMP1 were significantly higher in SNK-6 cells than in controls (Fig. 1A). A similar result was observed for protein levels (Fig. 1B).

To evaluate the effect of LMP1 on NKTL development, LV-LMP1-shRNA transfection was performed. After the selection of positive cells, LMP1 mRNA and protein expression were detected by qRT-PCR and western blotting. A statistically significant decrease in LMP1 levels was observed after LMP1 shRNA transfection, compared with the NC group (Fig. 1C and D). Following transfection with LMP1 shRNA for 48 h, an obvious decrease in cell proliferation rate was observed, with a 23.0% inhibition of proliferation compared to control cells (Fig. 2A). Given that LMP1 knockdown attenuated SNK-6 cell proliferation, we next performed cell cycle analyses on SNK-6 cells transfected with LMP1 shRNA vs. a control vector. Further flow cytometry assay showed that in comparison to the NC group, cells transfected with LMP1 shRNA induced reproducible and highly significant G0/G1 arrest. The distribution of transfected cells in the cell cycle increased by 13.4±5.7% in the G0/G1 phase (P<0.01) and decreased by 21.3±7.2% in the S phase (P<0.01) compared with controls. These results suggest that G0/G1 arrest may be the mechanism through which LMP1 regulates SNK-6 cell growth (Fig. 2B).

We further analyzed whether LMP1 shRNA pretreatment could influence the chemotaxis of SNK-6 cells. SNK-6 cell migration and invasion were assessed using a Transwell assay. As shown in Fig. 2C and D, after transfection with LMP1 shRNA, the migration and invasive potential of SNK-6 cells was markedly reduced, by 73.8±8.2 and 42.3±7.1% compared with the controls, respectively.

The apoptosis of differently treated SNK-6 cells was measured by FCM. The results showed that in parallel with the control groups, there was a significant increase in apoptosis, with LMP1 shRNA increasing cell apoptosis by 25.9±6.2% (Fig. 2E). This result indicates that LMP1 may inhibit cellular apoptosis in NKTL.

Increased eIF4E expression is associated with enhanced invasion and metastasis in many kinds of tumors (ref.?). To clarify the underlying mechanism involved in LMP1 regulated SNK-6 cell proliferation, migration, invasion and apoptosis, the expression of eIF4E was analyzed. As expected, after transfection with pcDNA3.0 or pcDNA3.0-LMP1 for 48 h, qRT-PCR analysis showed that compared with the control group, pcDNA3.0-LMP1 transfection increased the mRNA level of eIF4E by ~1.5-fold (Fig. 3A). Further analysis of LMP1 and eIF4E protein expression by western blotting showed that along with the increased protein level of LMP1, eIF4E expression was also upregulated compared with controls, suggesting that LMP1 tended to increase eIF4E expression (Fig. 3B and C).

To further analyze whether LMP1 regulated SNK-6 cell function via eIF4E expression, we performed a series of functional restoration assays. Following pretreatment with eIF4EshRNA, cells were then transfected with pcDNA3.0-LMP1, and the corresponding effect on cell proliferation, migration, invasion and apoptosis was assessed as before. The results showed that eIF4E silencing decreased the proliferation (Fig. 4A), migration (Fig. 4B), and invasion (Fig. 4C) of SNK-6 cells induced by LMP1 upregulation. The inhibitory effect on apoptosis triggered by LMP1 overexpression was also ameliorated in the eIF4E-silenced groups (Fig. 4D and E). These results suggested that LMP1 may trigger the proliferation, migration, invasion and apoptosis of SNK-6 cells by increasing eIF4E expression.

To further explore the underlying mechanism involved in LMP1-induced eIF4E expression, the NF-κB pathway was investigated. As expected, LMP1 overexpression clearly induced activation of the NF-κB pathway (Fig. 5A). However, following pretreatment with pyrrolidinedithiocarbamate (PDTC; Sigma), an inhibitor of the pathway, the expression of eIF4E induced by LMP1 was dramatically downregulated. Similar effects on eIF4E protein levels were observed, indicating that LMP1 induces eIF4E expression by activating the NF-κB pathway (Fig. 5B). SNK-6 cell proliferation, migration, invasion, and apoptosis were also assessed in order to certify whether the effect of LMP1 on SNK-6 cell function was mediated by the NF-κB pathway. MTT analysis showed that after pretreatment with PDTC, the proliferation induced by LMP1 was effectively attenuated (Fig. 5C). Moreover, the results of the transwell assay indicated that the changes in cell migration (Fig. 5D) and invasion (Fig. 5E) were consistent with proliferation. PDTC pretreatment enhanced cell apoptosis significantly compared with the LMP1 group (Fig. 5F). The results demonstrated that eIF4E is responsible for LMP1-induced SNK-6 cell proliferation, migration, invasion and apoptosis via the NF-κB signaling pathway.