EIF4G1 Was Highly Expressed in NPC Tissues and Cell Lines

We first set out to confirm our previous observation that EIF4G1 was a differentially expressed gene between normal and cancerous nasopharyngeal tissues. Using real-time PCR to measure the expression of EIF4G1 transcripts, we found that EIF4G1 expression was significantly increased in freshly isolated NPC tissues (n = 10) and NPC cell lines (n = 6) in comparison to freshly isolated nasopharyngeal tissues (n = 7) and the immortalized human nasopharyngeal epithelial cell lines NP69 (Figure 1A) (P< 0.001). Among the 6 NPC cell lines, 5-8F cells had the highest expression levels of EIF4G1 (Figure 1A); this cell line is also highly tumorigenic and metastatic [17, 18], suggesting of 5-8F cells as a good model system for studying the functions of endogenous EIF4G1 by loss-of-function approach.

Relationship between Clinicopathological Characteristics and EIF4G1 Expression in Patients with NPC

The relationship between clinicopathological characteristics and EIF4G1 expression in patients with NPC is summarized in Table 1. We did not find any significant association of EIF4G1 expression with age, sex, smoking status, or distant metastasis (M classification) in 132 patients with NPC. However, we observed that the levels of EIF4G1 expression were closely correlated with the T classification (T1-T2 vs. T3-T4) (P = 0.039), lymph node involvement (N classification, N0-N1 vs. N2-N3) (P = 0.008), and clinical stage (I-II vs. III-IV) (P = 0.003) in NPC patients.

Reduced EIF4G1 Expression Suppressed the Proliferation, Cell Cycle Progression, Migration, and Invasion of NPC Cells in vitro

To study the biological function of EIF4G1, we used a lentiviral vector containing shRNA specifically targeting EIF4G1 to stably knock down the endogenous expression of EIF4G1 in 5-8F cells, an NPC cell line with high levels of endogenous EIF4G1. As shown in Figure 2A, comparing to the control (shRNA-Ctrl), cells transfected with shRNA-EIF4G1 had significantly decreased levels of EIF4G1 protein.

Subsequently, we examined the effect of decreased EIF4G1 expression on NPC cell growth in vivo. Using MTT assay, we found that the parental NPC 5-8F cells had a similar growth rate as shRNA-Ctrl cells over a seven-day period, while starting from day 2 the growth of shRNA-EIF4G1 cells was significantly slower than the former two cells (P < 0.05) (Figure 2B). Interestingly, the consistent result also appeared in the plate clone formation test. Both the parental 5-8F cells and the shRNA-Ctrl cells formed a similar number of colonies on plate over a two-week period [(73 ± 3.6) vs. (74.3 ± 4.93)]. In contrast, knocking down endogenous EIF4G1 could dramatically reduce the number of colonies (40 ± 5.0) (P < 0.05) (Figure 2C).

We also measured the alteration of cell cycle progression by EIF4G1 knock-down. Using flow cytometry analysis, we found that EIF4G1-deficient cells showed a significant increase in S phase proportion and a decrease in G2/M proportion compared to the shRNA-Ctrl and the parental 5-8F cells (P < 0.05) (Figure 3A).

Cell migration and invasion are integral steps for the process of tumor development and metastasis. When testing the abilities of 5-8F cells to migrate/invade through the 8-μm pores on the polycarbonate membrane with or without pre-coated matrigel, we found that the knock-down of endogenous EIF4G1 expression could significantly reduce cell migration and invasion as compared to the parental or shRNA-Ctrl cells (P < 0.05) (Figures 3B, C).

Knock-down of EIF4G1 Inhibited in vivo Xenograft Tumor Growth

In addition to examining the biological functions of EIF4G1 in vitro, we also assessed the in vivo function of EIF4G1 in a xenograft tumor transplantation model. After subcutaneously transplanting the cells containing shRNA-Ctrl or shRNA-EIF4G1 lentiviral vectors into nude mice, we monitored the tumor growth over a 25-day period. As shown in Figure 4A, by measuring the tumor weights, we found that shRNA-EIF4G1 cells gave rise to significantly smaller tumors than shRNA-Ctrl cells did (P < 0.05). Immunohistochemistry assay revealed that protein expression of EIF4G1 was significantly decreased in the tumors induced by shRNA-EIF4G1 cells compared to those induced by shRNA-Ctrl cells (Figure 4B), which was in consistent with the in vitro results in cells transfected with shRNA-EIF4G1 or the control vectors (Figure 2A).

EIF4G1 Inhibited the Expression of PDCD4 in NPC cells

The above results indicated that over-expression EIF4G1 may play an important role in promoting the development of NPC. We further examined the effect of EIF4G1 on the expression of PDCD4, a key tumor suppressor protein interacting with EIF4G1 and controlling tumor growth and invasion [19]. We speculated that reducing the levels of EIF4G1 activates the effects of PDCD4. To test this hypothesis, we measured the protein levels of PDCD4 in cells deficient of EIF4G1. Comparing to the parental 5-8F cells and cells containing the control vector, EIF4G1-deficient cells had increased levels of PDCD4 protein (Figure 5A). Our results suggest that EIF4G1 may be involved in the development of NPC by antagonizing the effect of PDCD4.

Cell Culture and Sample Collection

NPC cell lines 5-8F, 6-10B, CNE2, HONE1, HNE1, and SUNE1 were maintained in RPMI 1640 culture medium supplemented with 10% newborn calf serum (NBCS) (PAA Laboratories, Austria). NP69, an immortalized human nasopharyngeal epithelial cell line, was grown in defined-KSFM medium supplemented with EGF (Invitrogen, Carlsbad, CA). All of the cell lines were incubated in a humidified chamber with 5% CO₂ at 37°C. Seven primary NPC tissues, 5 noncancerous nasopharyngeal tissues, 132 paraffin-embedded NPC specimens (107 cases with prognosis information), and 53 noncancerous paraffin-embedded nasopharyngeal specimens were obtained from People's Hospital of Zhongshan City, China, at the time of diagnosis before any therapy. An informed consent was obtained from each patient. All freshly collected samples were immediately stored in liquid nitrogen.

Real-time PCR

Real-time PCR was performed to measure the expression of EIF4G1 mRNA in NPC cell lines, tumor tissues, or normal nasopharyngeal tissues using SYBR Premix Ex Taq (Takara, Japan) as described previously [28]. The primers used were: forward 5'-TTGTGGATGATGGTGGCT-3' and reverse 5'-TTATCTGTGCTTTCTGTGGGT-3'. GAPDH was used as an internal control and the primers were: forward 5'-GCACCGTCAAGGCTGAGAAC-3' and reverse 5'-TGGTGAAGACGCCAGTGGA-3'. Each measurement was repeated three times.

Immunohistochemistry

Paraffin sections (3 μm) from 132 NPC and 53 nasopharyngeal samples were deparaffinized in 100% xylene and re-hydrated in descending ethanol series according to standard protocols [7]. Heat-induced antigen retrieval was performed in 10 mM citrate buffer for 2 min at 100°C. The endogenous peroxidase activity and non-specific antigens were blocked with peroxidase blocking reagent containing 3% hydrogen peroxide and serum. The slides were incubated with rabbit anti-human EIF4G1 antibody (1:50) (Cell Signaling Technology, CA, USA) for 1 h at 37°C. After washing, the sections were incubated with biotin-labeled goat anti-rabbit IgG antibody for 10 min at room temperature followed by incubation with streptavidin-conjugated horseradish peroxidase (HRP) (Maixin Inc, China). The peroxidase reaction was developed with 3,3-diaminobenzidine chromogen solution in DAB buffer substrate. The slides were reviewed and scored independently by two pathologists blinded to the clinical parameters. The staining intensity was scored as described [29, 30]. The extent of the staining, defined as the percentage of positive staining cells in relation to the whole field, was scored on a scale of 0 to 3 as 0: < 10%; 1: 10-25%; 2: 26-75%; and 3: ≥ 76%. The sum of the staining-intensity and staining-extent scores (0-6) was used as the final staining score for EIF4G1. For statistical analysis, a final staining score of 0, 1~2, 3~4, and 5~6 were respectively considered to be negative, low, medium, and high expression levels.

Establishment of NPC 5-8F cell line with stably expressing shRNA-EIF4G1

The construction of lentiviral vector of shRNA targeting EIF4G1 and establishment of NPC 5-8F cell line stably expressing shRNA had been described previously [17, 18, 31]. In brief, we selected one sequence for targeting the EIF4G1 gene using the BLOCK-It RNAi Designer (Invitrogen, Carlsbad, CA). The preparation of lentiviral vectors expressing human EIF4G1 short hairpin RNA (shRNA) was performed using the BLOCK-It Lentiviral RNAi Expression System (Invitrogen, Carlsbad, CA), following the manufacturer's instruction and replication-incompetent lentivirus was produced by cotransfection of the pLenti6/EIF4G1 expression vector and ViraPower packaging mix (Invitrogen) containing an optimized mixture of three packaging plasmids: pLP1, pLP2, and pLP/VSVG into 293FT cells. NPC 5-8F cells were infected with lentiviral particles containing specific or negative control vectors and selected for stable integrants by culturing a complete medium containing blasticidin. After 15 days of selection, there were no viable cells in mock wells and discrete blasticidin resistance colonies. The total RNA of these cell clones was isolated, and the levels of EIF4G1 mRNA were measured using real-time PCR as described above.

Western blot Analysis

Cells were lysed in RIPA Buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 5 mM DTT, 2% SDS), and protein concentration was determined using BCA assay (Beyotime Inc, China). Total protein (30 μg) was resolved using a 10% SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. The primary antibodies used were rabbit polyclonal anti-EIF4G1 antibody(1:500), anti-ACTB antibody(1:400) (both from Santa Cruz Biotechnology, CA, USA) and PDCD4(1:500, Abcam Inc., Cambridge, MA). An HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody (Zhongshan Inc, China).

Cell Proliferation and Cell Cycle Analyses

Cell proliferation was analyzed using MTT assay (Sigma, St. Louis, USA). Briefly, 1 × 10³ cells were seeded into a 96-well plate with quadruplicate repeat for each condition. After 72 h of incubation, MTT reagent was added to each well and incubated for 4 h. The formazan crystals formed by viable cells were then solubilized in DMSO and measured at 490 nm for the absorbance (A) values. Each experiment was performed in triplicates.

To evaluate cell cycle distribution, cells were seeded on 10 cm-diameter plates in RPMI 1640 culture medium containing 10% NBCS. After 48 h of incubation, a total of 1 × 10⁶ cells were harvested, rinsed with cold PBS, and fixed with 70% ice-cold ethanol for 48 h at 4°C. Fixed cells were rinsed with cold PBS followed by incubation with PBS containing 10 μg/mL propidium iodide and 0.5 mg/mL RNase A for 15 min at 37°C. The DNA content of labeled cells was acquired using FACS Caliber cytometry (BD Biosciences). Each experiment was performed in triplicates.

In vitro Cell Migration and Invasion Assays

Cells growing in the log phase were treated with trypsin and re-suspended as single-cell solution. A total of 1 × 10⁵ cells were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning Inc., Corning, NY). In the lower chamber, 600 μl of RPMI 1640 with 10% NBCS was added as chemoattractant. After the cells were incubated for 12 h, the insert was washed with PBS, and cells on the top surface of the insert were removed by a cotton swab. Cells adhering to the lower surface were fixed with methanol, stained with Giemsa, and counted under a microscope in five predetermined fields (×200). For the Matrigel invasion assay, the procedure was similar with the cell migration assay, except that the transwell membrane was precoated with ECMatrix™ and the cells were incubated for 14 h. All assays were independently repeated at least three times.

Colony Formation Assay

About 100 cells were added to each well of a 6-well culture plate, and each cell group contained 2 wells. After 2 weeks of incubation, cells were washed twice with PBS and stained with Giemsa solution. The number of colonies containing ≥ 50 cells was counted under a microscope. The colony formation efficiency was calculated as: efficiency = (number of colonies/number of cells inoculated) × 100%. Each experiment was performed in triplicates.

Growth of Tumor Xenografts in Nude Mice

All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Animals under assurance number A3873-1. Nude mice of 4 to 6 weeks old (n = 6) were purchased from the Animal Center, Southern Medical University. A total of 1 × 10⁶ cells growing in log phase were re-suspended in serum-free RPMI 1640 medium and injected subcutaneously into the nude mice. To minimize inter-individual variation, cells containing control vector and cells containing shRNA-EIF4G1 vector were injected symmetrically to the either flank of the same mice. After 25 d, mice were sacrificed and tumors isolated and weighted. EIF4G1 protein levels in the xenograft tumors were examined using immunohistochemistry assay.

Statistical analysis

All data were analyzed for statistical significance using SPSS 13.0 software. The Mann-Whitney U test was applied to the analysis of relationship between EIF4G1 expression levels and clinicopathologic characteristics. Survival analysis was performed using Kaplan-Meier method (the log-rank test). Multivariate Cox proportional hazards method was used for analyzing the relationship between the variables and patient's survival time. One-way ANOVA was used to determine the differences between groups for all in vitro and in vivo analyses. A P value of less than 0.05 was considered statistically significant.