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Keywords:

  • NESG1;
  • nasopharyngeal carcinoma;
  • tumor suppressor

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Human NESG1 (CCDC19) gene was originally isolated in our laboratory from human nasopharynx tissue. However, the biological and clinical significances of this gene remain largely unknown. In this report, two errors in the originally submitted sequence of human NESG1 gene were found, and the open reading frame sequence of NESG1 (Accession number: NM_012337.1) was revised and updated in the NCBI database (Accession number: NM_012337.2). The antibody raised against the revised sequence of NESG1 detected a single band of 66 kD in human nasopharynx tissues. NESG1 transcripts were specifically expressed in the nasopharynx epithelium. Expression of NESG1 transcripts and protein was downregulated or absent in nasopharyngeal carcinoma (NPC) tissues and cell lines in comparison to that in the normal nasopharynx tissues. The levels of NESG1 protein were significantly greater in the low-grade NPC tissues than that in the high-grade NPC tissues. Induced expression of NESG1 in otherwise NESG1-negative 5-8F cells not only significantly decreased cell proliferation, G1-S phase transition, but also markedly inhibited the ability of cell migration and invasion as well as in vivo tumorigenesis. Furthermore, NESG1 also significantly regulated the expression of cell cycle regulator CCNA1 and p21. Our findings first provided evidence that NESG1 may act as a tumor suppressor by inhibiting cell proliferation, invasion and migration of NPC cells.

Nasopharyngeal carcinoma (NPC), a disease in which malignant cells form in the tissues of the nasopharynx, is one of the most common malignancies in Southern China and Southeast Asia. It poses one of the serious health problems in southern China where an annual incidence of more than 20 cases per 100,000 is reported. Men are twice as likely to develop NPC as women. The rate of incidence generally increases from ages 20 to around 50.1 Synergetic effects of virus infection, genetic alteration and environmental factors are believed to cause the abnormal gene expression, which contributes to the development of NPC. Among these changes, the activation of oncogenes and inactivation of tumor suppressor genes may be key steps for initiating tumor formation and development. Epstein-Barr virus encoded latent membrane protein 1 (LMP1) has been considered as an oncogenic protein for its transform and tumorigenic activities. Biologically, LMP1 is an integral membrane protein that facilitates self-aggregation in the plasma membrane and transduces ligand-independent signals, including NF-κB, c-Jun-NH2-terminal kinase (JNK), p38/MAPK, Ras/MEK/ERK/MAPK, PI3K/Akt, Op18/stathmin and JAK/STAT.2, 3 Although the fundamental function of LMP1 is to inhibit cell apoptosis, it has other important roles in carcinogenesis. NPC cells with LMP1-positive expression have greater mobility, leading to higher metastatic potential and faster disease progression.4–6LMP1 is also involved in suppressing immunogenic responses against NPC; for example, LMP1 suppresses T-cell activation7 and mediates downregulation of CD99,8 an important component of the anti-NPC immune response. B-lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), a polycomb gene family member that is considered as an oncogene, plays an important role in cell cycle regulation, cell immortalization and cell senescence. Recent study has demonstrated that Bmi-1 is dysregulated in various cancers,9, 10 and its upregulation strongly correlates with an invasive phenotype and poor prognosis in patients with NPCs. Furthermore, increased Bmi-1 expression can repress the tumor suppressor PTEN and induce epithelial-mesenchymal transition in human nasopharyngeal epithelial cells.11 Survivin, a member of the baculovirus inhibitors of apoptosis gene, correlates not only with apoptosis resistance but also with the regulation of cell proliferation and angiogenesis in cancer.12, 13 Elevated expression of survivin has been indicated in NPC, which is an unfavorable prognostic factor for the survival of NPC patients.14RASSF1A residing on chromosome 3p21, a region frequently found inactivated in human cancers, is revealed as a tumor suppressor gene in NPC by allelic deletion and promoter methylation.15 Downregulated expression of RASSF1A showed a significant negative association with WHO grade, tumor status and lymph node metastasis in NPC.16 High frequency of mutations in the TP53 gene, a well-characterized tumor suppressor, has been documented to be associated with a variety of human malignancies.17, 18 However, in NPC the frequency of TP53 mutation is low, suggesting the unimportance of inactivation of TP53 in NPC development.19 In the past few years, although the knowledge of molecular basis about NPC has greatly increased, the pathogenesis of NPC still remains to be fully investigated. Thus, the discovery and identification of NPC-associated genes would help further elucidate the molecular mechanism of NPC.

Previously, we studied the differential gene expression between normal human nasopharynx mucosa and oral cavity mucosa of soft palate using gene differential display, and found a full-length NESG1 gene specifically expressed in human nasopharynx and trachea.20 The cloned NESG1 gene had an open reading frame (ORF) of 1,161 nucleotides encoding a soluble basic protein of 386 amino acids (Accession number NM_012337.1). However, little, if any, information has since been generated about the structure of this gene. Moreover, the biological function and clinical significance of NESG1 have remained largely unknown. In this study, we set out to reclone NESG1 gene in human nasopharynx tissue and to investigate the function and clinical significance of NESG1 in NPC. We identified two errors in the sequence of original submitted version of NESG1, and found that NESG1 can inhibit cell proliferation, invasion and migration of NPC cells.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Cell culture and sample collection

Eight NPC cell lines including 5-8F, 6-10B, CNE2, CNE1, C666-1, HONE1, HNE1 and SUNE1 were maintained in RPMI 1640 supplemented with 10% newborn calf serum (NBCS) (PAA Laboratories, Austria). Twelve freshly isolated primary NPC tissues, ten freshly isolated non-cancerous nasopharynx tissues, 82 paraffin-embedded undifferentiated NPC specimens and 40 paraffin-embedded non-cancerous nasopharynx specimens were obtained at the time of diagnosis before any therapy from People's Hospital in Zhongshan City, China. An informed consent was obtained from each patient.

Recloning of NESG1 and sequence verification

The total RNA of three non-cancerous nasopharynx tissues was extracted and cDNA was reverse-transcribed from 1 μg of total RNA using oligo(dT)18 in 20 μl reaction volumes. The original coding region of NESG1 (Accession number NM_012337.1) was amplified using specific primers: forward 5′-CGGAATTCATGGATGCAGTGATGACACGA-3′; reverse 5′-ATTGGTACCAAGCTCTTCAAGCTTTTTCCTCT-3′. The PCR product digested by restriction endonucleases EcoRI and KpnI was then ligated into pEGFP-N1 vector that was digested with the same restriction endonucleases. The plasmid insert was sequenced, and the sequence was compared with the original NESG1 sequence, human genome sequences and NCBI nucleotide sequence database using NCBI's BLAST tool (Basic Local Alignment Search Tool).

Antibody preparation and western blotting assay

The ORF of revised NESG1 was amplified from a plasmid containing NESG1 full-length sequence (IMAGE: 30387473) obtained from Invitrogen Incorporation (Carlsbad, CA), and was cloned into prokaryotic expression vector pGEX-4T-1. Subsequently, recombinant GST-NESG1 fusion protein expression was induced using IPTG, and the resulting protein was purified using GSH-sepharose. The purified protein was used to immunize rabbits. The antigenicity of this fusion protein was validated using western blotting assay with rabbit anti-GST antibody (Santa Cruz, CA; 1:3,000) followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Bio-Rad; 1:5,000).

Nasopharynx or NPC tissues were grinded in liquid nitrogen and lysed in RIPA buffer on ice containing protease inhibitors. Cells were directly lysed in RIPA buffer. Protein lysates were resolved on 10% SDS (sodium dodecyl sulfate) polyacrylamide gel, electro-transferred to polyvinylidene fluoride membranes (Invitrogen, Carlsbad, CA) and blocked with 5% nonfat dry milk in Tris-buffered saline, pH 7.5. Membranes were immunoblotted overnight at 4°C with anti-NESG1 polyclonal antibody or anti-β-actin antibody (Santa Cruz, CA), followed by their respective HRP-conjugated secondary antibodies. Signals were detected using enhanced chemiluminescence reagents (Pierce, Rockford, IL).

Localization of NESG1 expression in human nasopharynx mucosa

A digoxigenin-labeled cDNA probe for NESG1 (5′-TGCCGCTTCCAACAGGTCAA-3′) was used to examine the localization of gene expression in nasopharynx mucosa by chromogenic in situ hybridization (CISH) using reagent kits from Zymed Laboratories (South San Francisco, CA). Briefly, the sections were incubated for 1 hr at 59°C, and deparaffinized in xylene and graded ethanol. The sections were digested with trypsin for 30 min at 37°C, washed with PBS (phosphate buffered saline) and dehydrated in graded dilutions of ethanol. The NESG1 probe (10 μl/slide) was applied to the slides under coverslips. The slides were denatured on a hot plate for 3 min at 94°C, followed by overnight hybridization at 37°C. After stringent washing, the hybridized probe was detected with sequential incubation with mouse anti-digoxigenin, HRP-labeled goat anti-mouse IgG antibody, and DAB. Tissue sections were lightly counterstained with hematoxylin and mounted.

NESG1 mRNA measurement

Total RNA from one pooled nasopharynx sample and eight pooled NPC samples were used to examine the differential expression of NESG1 in human nasopharynx and NPC samples using semi-quantitative RT-PCR (reverse transcription-polymerase chain reaction).21 The RT-PCR results were normalized to the invariant housekeeping ACTG1 gene. Furthermore, we used real-time RT-PCR to measure the expression level of NESG1 in ten noncancerous nasopharynx samples, 12 NPC samples and eight NPC cell lines. ARF5, an invariant housekeeping gene in nasopharynx and NPC samples, was used as inner control. The primer sequences for NESG1 were: forward 5′-CGCCTGTGAGTGAGTGC-3′ and reverse 5′-CTTATCCATCCTTTCGGTCTT-3′. The primer sequences for ARF5 were: forward 5′-ATCTGTTTCACAG TCTGGGACG-3′ and reverse 5′-CCTGCTTGTTGGCAAA TACC-3′. The PCR reaction was carried out using SYBR Green Mix reagent (Takara, Japan). Both semi-quantitative RT-PCR and real time RT-PCR were repeated thrice.

Immunohistochemistry staining

Paraffin sections (3 μm) from samples of 82 undifferentiated NPC and 40 nasopharynx were deparaffinized with 100% xylene and rehydrated in descending percentage of ethanol series according to standard protocol.22, 23 Heat-induced antigen retrieval was performed in 10 mM citrate buffer for 2 min at 100°C. Endogenous peroxidase activity and nonspecific antigen were blocked with peroxidase blocking reagent containing 3% hydrogen peroxide and serum, followed by incubation with rabbit anti-human NESG1 antibody (10 μg/ml) for 1 hr at 37°C. After washing, the sections were incubated with biotin-labeled goat anti-rabbit IgG antibody for 10 min at room temperature and subsequently conjugated with HRP (Maixin, China). Sections were visualized with DAB (3,3′-Diaminobenzidine) and counterstained with hematoxylin, mounted in neutral gum and analyzed using a bright-field microscope. The stained tissue sections were reviewed and scored independently by two pathologists blinded to the clinical parameters. The staining intensity was scored as 0 (negative), 1 (weak), 2 (medium) and 3 (strong).24 The extent of the staining, defined as the percentage of positive staining areas in relation to the whole section area, was scored on a scale of 0–3 as 0 for <10%, 1 for 10–25%, 2 for 26–75% and 3 for ≥76%. The sum of the staining-intensity and staining-extent scores was used as the final staining score for NESG1. For statistical analysis, a final staining score of 0, 1–2, 3–4 and 5–6 were considered to be negative, low, medium and high expression, respectively.

Construction of pGC-FU-NESG1-GFP vector and lentivirus infection

The NESG1 ORF was amplified using the forward primer 5′-CAGGATCCCCGGGTACCGGTCGCCACCATGCCACTAAG CACAGCTG-3′ and the reverse primer 5′-TCACCATGGTGG CGACCGGTACGTTCACAGAGGTAGCTGGCA-3′ with the introduction of restriction endonuclease AgeI site. NESG1 cDNA digested with AgeI was cloned into an AgeI-digested pGC-FU-GFP lentivirus vector (Genechem Incorporation, Shanghai, China). The resulting lentivirus vector together with pHelper1 and pHelper2 vectors were co-transfected into 293FT cells for 84 hr using lipofectamine 2000 (Invitrogen, Carlsbad, CA) to generate lentiviral stock. An “empty” vector pGC-FU-GFP was utilized as a negative control. After the titers were determined, the lentiviral particles were used to infect NESG1-negative 5-8F cells, an NPC cell line with high-metastatic potential.25 Colonies with GFP expression were selected to expand culture for further investigation.

MTT assay

The rate of in vitro cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates at a density of 1,000 cells/well. The cells were incubated for 1, 2, 3, 4, 5, 6 or 7 days. Twenty microliters of MTT (5 mg/ml) (Sigma, St. Louis, MO) was added to each well and incubated for 4 hr. At the end of incubation, the supernatants were removed, and 150 μl of DMSO (dimethyl sulfoxide) (Sigma, St. Louis, MO) was added to each well. The absorbance value (OD) of each well was measured at 490 nm. For each experimental condition, eight wells were used. Experiments were performed thrice.

Colony formation assay

Cells were plated in 6-well culture plates at 100 cells/well. Each cell group had two wells. After incubation for 9 days at 37°C, cells were washed twice with PBS and stained with the Giemsa solution. The number of colonies containing ≥50 cells was counted under a microscope. The colony formation efficiency was calculated as (number of colonies/number of cells inoculated) × 100%.

Cell cycle analysis

For cell cycle analysis, cells were seeded on 10-cm–diameter plates in RPMI 1640 containing 10% NBCS. After incubation for 48 hr, a total of 5 × 106 cells were harvested, rinsed with cold PBS and fixed with 70% ice-cold ethanol for 48 hr 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 FACSCaliber cytometry (BD Biosciences). Each experiment was performed in triplicate.

Cell migration and invasion assays

For the cell migration assay, 1 × 105 cells in 100 μl RPMI 1640 medium without NBCS were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Costar, MA). In the lower chamber, 600 μl RPMI 1640 with 10% NBCS was added as chemoattractant. After the cells were incubated for 12 hr at 37°C in a 5% CO2 atmosphere, the insert was washed with PBS, and cells on the top surface of the insert were removed with a cotton swab. Cells adhering to the lower surface were fixed with methanol, stained with Giemsa solution and counted under a microscope in five predetermined fields (200×). All assays were independently repeated at least thrice.

For the cell invasion assay, the procedure was similar to the cell migration assay, except that the transwell membranes were precoated with 24 μg/μl Matrigel (R&D Systems, USA) and the cells were incubated for 16 hr at 37°C in a 5% CO2 atmosphere. Cells adhering to the lower surface were counted the same way as for the cell migration assay.

In vivo tumorigenesis in nude mice

A total of 1 × 106 logarithmically growing 5-8F cells transfected with pGC-FU-GFP-NESG1 and the control pGC-FU-GFP vector (3D8/C6, N = 6; 2F4/C6, N = 5) in 0.1 ml RPMI 1640 medium were subcutaneously injected into the left-right symmetric flank of 4–6-week-old male BALB/c nu/nu mice. The mice were maintained in a barrier facility on HEPA-filtered racks. The animals were fed with an autoclaved laboratory rodent diet. All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals under assurance number A3873-1. After 14 days and 18 days, the mice of 3D8/C6 and 2F4/C6 were, respectively, sacrificed. Tumor tissues were excised and weighted.

Microarray analysis

Total RNA from six samples, including two from NESG1-negative C6-Ctr cells, two from NESG1-expressing 2F4 cells and two from NESG1-expressing 3D8 cells, were prepared using RNeasy kit (Qiagen, Chatsworth, CA). All of these three types of cells were derived from the NESG1-negative 5-8F cells; 2F4 and 3D8 clones were introduced NESG1 expression using lentivirus containing NESG1 gene, and C6-Ctr was a NESG1-negative clone. Gene expression profiles were determined using Affymetrix Human Genome U133 Plus 2.0 Array containing 47,000 transcripts (Affymetrix, Santa Clara, CA). Hybridization, washing, staining, and scanning of GeneChips were performed according to the standard Affymetrix protocol. Significance analysis of microarray (SAM) was used to identify genes differentially expressed in 2F4, 3D8 and C6-Ctr cells.

mRNA and protein measurements of cell cycle regulators

The mRNA levels of cell cycle regulators CCNA1, CCND1, CDK4, CDK2, CDC2 and p21 in C6-Ctr, 3D8 and 2F4 cells were measured using real-time RT-PCR. The protein levels of these cell cycle regulators were determined using western blotting analysis. The antibodies used in this study were as following: CCNA1 (ab53699, Abcam, Landon), CCND1, CDK4 and p21 (Santa Cruz, CA).

Statistical analysis

SPSS 13.0 software (SPSS, North Chicago, IL) was used for statistical analysis. Mann-Whitney U-test and Kruskal-Wallis test were used to analyze the relationship between NESG1 expression and clinicopathologic characteristics of NPC patients. Analysis of variance (ANOVA) was used to test the significance of the effect of NESG1 on cell proliferation, migration, invasion and colony formation. T-test was used to examine the significance of the effect of NESG1 on in vivo tumorigenesis in nude mice. Differences were considered statistically significant when p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Expression of NESG1 gene in NPC tissues and cell lines

After updating the ORF sequence of NESG1 (Supporting Information Fig. SA), we localized the expression of NESG1 in human nasopharynx tissue by performing in situ hybridization assays. Figure 1a shows a strong expression of NESG1 in human nasopharynx epithelial cells. Subsequently, we used semi-quantitative RT-PCR and reverse transcription PCR to examine NESG1 expression in eight NPC cell lines and eight pooled NPC tissues. When comparing with normal nasopharynx tissues, we found that NESG1 expression was downregulated or absent in NPC tissues (Fig. 1b1) and undetectable in all eight NPC cell lines (Fig. 1b2). We further used real-time PCR to quantitatively measure NESG1 expression in 12 NPC samples, eight NPC cell lines, and ten normal nasopharynx tissue samples. The results showed that NESG1 expression was significantly decreased in all NPC cell lines and NPC tissues comparing to that in normal nasopharynx tissues (Fig. 1b3).

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Figure 1. Expression of NESG1 in NPC tissues, NPC cell lines and noncancerous nasopharynx tissues. (a) In situ hybridization showed that NESG1 was expressed in nasopharynx epithelial cells. (b and c) mRNA and protein expression of NESG1 were downregulated or lost in nasopharyngeal carcinoma tissues comparing to noncancerous nasopharynx tissues by using semiquantitative RT-PCR, RT-PCR, qRT-PCR and immunohistochemistry assay (×200).

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Using the newly prepared anti-NESG1 rabbit polyclonal antibody, we investigated NESG1 protein expression in nasopharynx and NPC tissues. In normal nasopharynx tissue, western blotting assay showed a protein band with apparent molecular weight of ∼ 66 kD (data not shown), which is in agreement with the calculated molecular weight based on the revised NESG1 ORF sequence. Immunohistochemistry staining was performed for 40 normal nasopharynx tissues and 82 NPC tissues. As shown in Figure 1c, NESG1 protein was abundantly expressed in nasopharynx epithelial cells and mainly localized in cytoplasm. In contrast, NESG1 expression was downregulated in NPC tissues (p < 0.001) (Table 1).

Table 1. Relationship between clinicopathologic characteristics and NESG1 expression in patients with NPC
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Relationship between clinicopathologic characteristics and NESG1 expression in patients with NPC

The relationship between clinicopathologic characteristics and NESG1 expression in patients with NPC is summarized in Table 1. There was no significant association of NESG1 expression with patients' age and gender. However, we found that NESG1 expression was closely correlated with patients' clinical stage (based on the 1997 UICC Staging System for NPC). The levels of NESG1 protein were significantly greater in NPC patients at early stages (I and II) than that in NPC patients at advanced stages (III and IV) (p = 0.001). In addition, based on TNM classification, we found that NESG1 protein levels were greater in NPC patients at N1-N2 than that in NPC patients at N3-N4 (p = 0.035) (Table 1).

NESG1 inhibits cell proliferation, migration and invasion

To study the biological functions of NESG1, we used a NESG1-negative NPC cell line 5-8F and introduced NESG1 into these cells using lentivirus containing NESG1 gene. Four stably transfected cell clones were obtained (2F4, 3G6, 3A6 and 3D8) (Fig. 2a). Western blotting assay showed that NESG1 protein expression in 2F4 and 3D8 cells was markedly increased compared with NESG1-negative clone C6-Ctr cells (Fig. 2b). The expression level of NESG1 in 2F4 cells was 1.76-fold higher than that in 3D8 cells (Fig. 2c).

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Figure 2. Stable expression of exogenous NESG1 cDNA in NPC cell lines. (a) NESG1 was highly re-expressed in clone 2F4, 3G6, 3A6 and 3D8 detected by RT-PCR after infection and selection. (b) Restored protein expression of NESG1 was displayed in 2F4 and 3D8 cells by western blot detection. (c) Bar graph showed the differences of NESG1 protein expression among the three groups. Data were presented as mean ± SD for three independent experiments. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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To analyze the function of NESG1, we studied the rate of cell proliferation of NESG1-expressing 2F4 and 3D8 cells. The growth curves determined by MTT assay showed that NESG1 significantly inhibited cell proliferation of these two lines of cells in comparison with NESG1-negative parental line 5-8F and clone C6-Ctr cells (Fig. 3a). The results from colony formation assay showed that NESG1-expressing 2F4 and 3D8 cells formed significantly less colonies than NESG1-negative C6-Ctr cells (p < 0.001 for both cell types) (Fig. 3b), suggesting the inhibitory effect of NESG1 on anchorage-dependent growth of NPC cells.

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Figure 3. Re-expression of NESG1 suppressed cell proliferation in vitro and tumorigenicity in vivo. (a and b) In vitro proliferative ability of NPC cells was significantly inhibited in NESG1-expressing 3D8 and 2F4 clones compared with NESG1 negative C6 clone by MTT assay and plate clone assay. Data were presented as mean ± SD for three independent experiments (*p < 0.05). (c) When compared with negative C6 clone, tumorigenicity of 3D8 and 2F4 cells was markedly reduced in vivo (*p <0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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To confirm the growth suppressive effect of NESG1, we performed in vivo tumorigenesis study by inoculating NESG1-expressing 2F4 and 3D8 cells into nude mice. The results showed that the mice of 3D8/C6-Ctr cell group were sacrificed 14 days after inoculation, the average weight of tumors was 0.297 g and 0.555 g, respectively (p = 0.005). In 2F4/C6-Ctr cell group, the average weight of tumors was respectively 0.288 g and 0.934 g (p < 0.001) when mice was sacrificed 18 days after inoculation (Fig. 3c). These results suggested a significant inhibitory effect of NESG1 on tumorigenesis in vivo.

To examine the effect of NESG1 on cell migration, NESG1-expressing 2F4 and 3D8 cells were cultured on transwell apparatus. After 12-hr incubation, the percentage of migrated cells was significantly less in both 2F4 and 3D8 cell groups than that in the parental 5-8F cells and the NESG1-negative C6-Ctr cells (for both p < 0.001) (Fig. 4a). Using a boyden chamber coated with matrigel, we determined changes in cell invasiveness after 16-hr incubation. When compared with the negative control 5-8F and C6-Ctr cells, NESG1-expressing 2F4 and 3D8 cells both showed significantly decreased invasiveness (for both p < 0.001) (Fig. 4b).

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Figure 4. Restoration of NESG1 expression inhibited cell migration, invasion and cell cycle progression in vitro. (a and b) Overexpressed NESG1 dramatically reduced the ability of 3D8 and 2F4 cell migration and invasion in vitro. (c) Overexpressed NESG1 retarded the cell cycle progression from G1 to S phase in 2F4 cells. Data were presented were presented as mean ± SD for three independent experiments (*p < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Inhibition of cell cycle by NESG1

To determine the effect of NESG1 on cell cycle, we first measured cell cycle distribution in NESG1-expressing 2F4 and 3D8 cells. The results showed that the S-phase population decreased significantly in 2F4 cells in comparison with the NESG1-negative C6-Ctr cells (p = 0.003). However, in 3D8 cells the change in S-phase population was not significant (Fig. 4c). This might be associated with the fact that 2F4 cells had higher levels of NESG1 than 3D8 cells did.

To further study the mechanism by which NESG1 regulates cell cycle, we examined the transcripts and protein levels of several key regulators of cell cycle (Supporting Information Table S). Using microarray analysis, we found that CCNA1 transcripts were markedly decreased in both NESG1-expressing 2F4 and 3D8 cells; however, the levels of p21 transcripts were increased only in 2F4 cells but not in 3D8 cells. The microarray results were verified using real-time PCR assays (Fig. 5a). Using western blotting analysis, we also found that CCNA1 protein levels decreased in both 2F4 and 3D8 cells; however, p21 protein levels only increased in 2F4 cells but not in 3D8 cells (Fig. 5b). Again, the discrepancy between 2F4 and 3D8 cells might be due to the fact that 2F4 cells express higher levels of NESG1 than 3D8 cells do. These results suggested a mechanism for the inhibitory effect of NESG1 on cell cycle.

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Figure 5. Decreased levels of CCNA1 but increased levels of p21 expression. (a and b) Restored NESG1 expression significantly inhibited the endogenous mRNA and protein expression of CCNA1 in 3D8 and 2F4 cells and activated the expression of p21 in 2F4 cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Before the completion of Human Genome Project,26, 27 some gene sequences were submitted to the Genbank database with various errors partially due to unavailable validation using human genome sequence. NESG1, a nasopharyngeal epithelium-specific gene, was isolated from human normal nasopharynx mucosa using improved differential display approach, and its sequences was submitted to the Genbank database by our research group in 1999.20 To verify the previous cloning results, in this study we recloned NESG1 gene and analyzed its sequence. Using NCBIs BLAST tool, we checked the revised sequences against the previous one, the human genome sequence and NCBIs nucleotide sequence database. We found that two errors in the originally submitted sequence lead to the alteration of ORF sequence of NESG1. The predicted ORF of the revised sequence is 1,656 bp with 11 exons encoding a protein of 551 amino acids, which extends 165 amino acids from the previously submitted NESG1 coding region. Also, the size of revised NESG1 ORF is the same as that of Macaca fascicularis.

In the previous study, NESG1 mRNA expression was detected in nasopharynx mucosa using northern blotting analysis.20 However, the exact localization of NESG1 expression in the stroma or epithelium cells of nasopharynx still remained to be determined. In this study, we confirmed the specific expression of NESG1 in nasopharyngeal epithelium cells using in situ hybridization, suggesting that NESG1 might play an important role in maintaining physiological functions of nasopharyngeal epithelium.

To identify the expression of NESG1 protein in nasopharynx mucosa tissue, we used newly prepared anti-NESG1 rabbit polyclonal antibody to examine its expression by using western blotting. A protein band with an apparent molecular weight of ∼66 kD was revealed, which is in agreement with the calculated molecular weight based on the revised NESG1 ORF sequence. Our result demonstrated the NESG1 gene was revised correctly by us.

Subsequently, we evaluated NESG1 mRNA expression in pooled nasopharynx tissues, NPC tissues and eight individual NPC cell lines using semi-quantitative RT-PCR. Interestingly, NESG1 expression was found to be downregulated or absent in NPC tissues and totally lost in all eight NPC cell lines. These results were quantitatively confirmed using real-time PCR. Furthermore, by using immunohistochemistry assays, we found that the protein levels of NESG1 were also markedly downregulated in NPC tissues comparing with the normal nasopharynx tissues. In addition, NESG1 expression levels were inversely associated with clinical stages. A low level of NESG1 seems to favor the development of NPC. These data strongly implied a suppressive role of NESG1 in NPC tumorigenesis in which loss of expression of NESG1 may promote NPC initiation and progression. If verifiable in a larger cohort of clinical patients, NESG1 could be used as a prognostic biomarker for NPC.

The biological functions of NESG1 found in this study provided a mechanistic basis for the pathological and clinical observations. The restored expression of NESG1 not only significantly inhibited the proliferation of NPC cells in vitro but also markedly suppressed tumorigenicity in an in vivo xenograft animal model.

This might be partly attributable to the suppressive effect of NESG1 on cell cycle. NESG1 can reduce the cell population at S-phase, suggesting an inhibition on the cell cycle progression from G1 to S phase. When we examined the key regulators of cell cycle at G1-S phase transition, we found that CCNA1 expression was inhibited whereas p21 was increased by NESG1. Interestedly, similar results were showed in our immunohistochemistry assays examining the expression of CCNA1 and p21 in transplantation tumor of C6, 3D8 and 2F4 cells (Supporting Information Fig. SB). CCNA1 over-expression has been found in several types of cancer, including prostate cancer, testicular germ cell tumors, and leukemia.28–30p21, a significant tumor suppressor,31 is a cyclin-dependent kinase (Cdk) inhibitor and involves in regulating cell cycle progression at the G1/S.32 The inhibitory effect on CCNA1 and the activated effect on p21 mediated by NESG1 suggest potential avenues to target CCNA1 and p21 protein in tumors. The suppressive effect of NESG1 on cell cycle is greater in 2F4 cells than in 3D8 cells. Considering the fact that 2F4 cells express higher levels of NESG1 than 3D8 cells do, these data suggest that the effect of NESG1 on cell cycle is dose-dependent, though a more thorough experiment is needed to verify this statement. Interestingly, unlike CCNA1 and p21, the expression levels of other significant cell cycle regulators including CCND1, CDK4, CDK2, CDC2,p27, p16, CCNA2, CCNE1, CCNE2 and Rb1 did not change in spite of the restored expression of NESG1. This suggests the uniqueness of regulatory effect of NESG1 on the cell cycle machinery in NPC cells. In addition to its effect on cell proliferation and cell cycle, NESG1 also inhibited the migration and invasiveness of NPC cells. Taken together, our results suggest that NESG1 is a tumor suppressor in the human nasopharynx. It would be interesting to find out whether NESG1 also exerts similar functions in other tissues.

In conclusion, this study revised the NESG1 gene sequence and, for the first time, revealed the biological functions and clinical significance of NESG1 in NPC. By virtue of the suppressive effect of NESG1 on NPC development, NESG1 has the potential to serve as a prognostic biomarker for NPC.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_25595_sm_suppfigSA.doc242KSupporting Figure 1
IJC_25595_sm_suppfigSB.doc1116KSupporting Figure 2
IJC_25595_sm_suppTable.doc45KSupporting Table

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