The 10 nasopharyngeal carcinoma tissues used in this study were obtained from the Taizhou People’s Hospital in China. Specimens were snap-frozen in liquid nitrogen. Fresh normal nasopharyngeal mucosal tissues were obtained from another 5 patients undergoing biopsies for other non-neoplastic diseases. The collection and use of the patient samples were reviewed and approved by the Institutional Ethics Committees and written informed consent from all patients was appropriately obtained.

mRNA expression profiling containing 31 nasopharyngeal carcinoma and 10 normal tissues samples (GSE12452) was carried out (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12452). Using the AmiGO tool (7) of the Gene Ontology project (8), lists of transcripts associated with the biological processes, proliferation (GO:0008283) and apoptosis (GO:0006915), were obtained. Lists of unique transcripts were prepared by removing duplicate entries. Subsequently, the microarray dataset was queried for the genes in each of these ontologies. Samples were sorted according to the EZH2 expression level for the nasopharyngeal carcinoma and 10 normal tissues samples separately and the average expression levels scaled on a gene by gene basis for genes significantly correlating with EZH2 expression (absolute value Pearson’s correlation >0.6) were plotted as a heatmap.

CNE-2 cells maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. Hsa-let-7a, siEZH2 and negative control oligonucleotides were purchased from GenePharma Co., Ltd. (Shanghai, China). For the expression plasmid construct, wild-type EZH2 cDNA sequence without 3’UTR was selected and cloned into the pGenesil-1 vector. Cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) at 50–60% confluency. Forty-eight hours after transfection, the cells were harvested for further experiments.

Real-time quantification of hsa-let-7a was performed by stem-loop RT-PCR. All the primers of miRNAs for TaqMan miRNA assays were purchased from GenePharma Co., Ltd. and were as follows: human EZH2 forward, 5′-CCTG AAGATGTCGGCATCGAAAGAG-3′ and reverse, 5′-TGCAA AAATTCACTGGTACAAAACACT-3′; and human GAPDH forward, 5′-GTCGGAGTCAACGGATT-3′ and reverse, 5′-AAG CTTCCCGTTCTCAG-3′. Real-time PCR was performed according to the manufacturer’s instructions. All experiments were performed using biological triplicates and experimental duplicates. The relative expression was calculated via the 2-ΔΔCt method.

Cells were plated at 10⁴ cells/well in 96-well plates with 6 replicate wells. After transfection as previously described, 20 μl of MTT (5 g/l, Sigma, St. Louis, MO, USA) were added into each well on 4 consecutive days after treatment and the cells were incubated for an additional 4 h. The supernatant was subsequently discarded. DMSO (200 μl) was added to each well to dissolve the precipitate. Optical density (OD) was measured at a wavelength of 550 nm. The data are presented as the means ± SD, which are derived from triplicate samples of at least 3 independent experiments.

Cells were washed with PBS and fixed with 70% ethanol for at least 1 h. After extensive washing, the cells were suspended in Hank’s balanced salt solution (HBSS) containing 50 μg/ml PI and 50 μg/ml RNase A and incubated for 1 h at room temperature and analyzed by FACScan (Becton-Dickinson, Franklin Lakes, NJ, USA). Cell cycle analysis was analyzed by ModFit software. Experiments were performed in triplicate. The results were presented as a percentage of cells in a particular phase.

Cells were plated into 6-well plates at 1×10⁵ cells/well. Forty-eight hours after transfection, the cells were harvested by trypsinization and washed with PBS. Annexin V and PI double-staining (BD Biosciences, San Jose, CA, USA) were used to detect and quantify cellular apoptosis by flow cytometry. Annexin V⁻ and PI⁻ cells were used as the controls. Annexin V⁺ and PI⁻ cells were designated as apoptotic; Annexin V⁺ and PI⁺ cells were considered necrotic. All the tests were performed in triplicate.

Equal amounts of protein per lane were separated by 8% SDS-polyacrylamide gel and transferred onto a PVDF membrane. The membrane was blocked in 5% skim milk for 1 h and then incubated with a specific antibody for 2 h. The antibodies used in this study were as follows: antibodies to EZH2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antibody against GAPDH (Santa Cruz Biotechnology) was used as the control. The specific protein was detected by using a SuperSignal protein detection kit (Pierce, Rockford, IL, USA). The band density of specific proteins was quantified after normalization with the density of GAPDH.

The human EZH2 3’UTR were amplified and cloned into the XbaI site of the pGL3-control vector (Promega, Madison, WI, USA), downstream of the luciferase gene, to generate the plasmids pGL3-WT-EZH2-3’UTR. pGL3-MUT-EZH2-3’UTR plasmids were generated from pGL3-WT-EZH2-3’UTR by deleting the binding site for let-7a ‘CUACCUC’. For the luciferase reporter assay, cells were cultured in 96-well plates, transfected with the plasmids and let-7a using Lipofectamine 2000. Forty-eight hours after transfection, luciferase activity was measured using the Luciferase assay system (Promega).

Statistics was determined by ANOVA, or the t-test using SPSS11.0. A value of P<0.05 was considered to indicate a statistically significant difference.

To explore let-7a expression in nasopharyngeal carcinoma, we examined 10 human nasopharyngeal carcinoma specimens and 5 normal nasopharyngeal mucosal tissues using real-time PCR. As shown in Fig. 1, the levels of let-7a decreased markedly in the nasopharyngeal carcinoma in comparison to the normal tissue samples (P<0.01).

By performing bioinformatics analysis of let-7a potential target genes, we found that the tumor suppressor EZH2 contained the highly conserved putative let-7a binding sites. To determine whether EZH2 is directly regulated by let-7a (Fig. 2A), western blot analysis and luciferase reporter assay were employed. Western blot analysis demonstrated a significant reduction in EZH2 expression following the overexpression of let-7a in the CNE-2 cells (Fig. 2B). Furthermore, we created the pGL3-WT-EZH2-3’UTR and pGL3-MUT-EZH2-3’UTR plasmids. Reporter assay revealed that the overexpression of let-7a triggered a marked decrease in the luciferase activity of the pGL3-EZH2-EZH2-3’UTR plasmid in the CNE-2 cells; however, no change in the luciferase activity of the pGL3-MUT-EZH2-3’UTR plasmid was observed (Fig. 2C).

We further explored the correlation between let-7a and EZH2 expression in nasopharyngeal carcinoma. We examined 10 human nasopharyngeal carcinoma specimens using real-time PCR. The levels of EZH2 increased markedly in the nasopharyngeal carcinoma in comparison to the normal tissues (P<0.02) (Fig. 3A). Additionally, Pearson’s correlation showed that a significant negative correlation existed between let-7a and EZH2 expression in the nasopharyngeal carcinoma tissues (R=-0.7992, P<0.01) (Fig. 3B). These data indicate that EZH2 is a direct target of let-7a in nasopharyngeal carcinoma.

To determine the EZH2-mediated effects of let-7a on nasopharyngeal carcinoma cell proliferation, we first analyzed which genes associated with cell proliferation correlated with EZH2 expression in nasopharyngeal carcinoma. First, EZH2 was found to be overexpressed in the majority of the nasopharyngeal carcinoma samples as compared to the normal tissues. However, in a few samples the EZH2 mRNA expression was found to be in the same range as in the normal tissues (Fig. 4A). Out of the 1,134 genes that were linked to cell proliferation as determined by AmiGO (7), 57 genes showed a clear correlation (>60%) with EZH2 expression in nasopharyngeal carcinoma (Fig. 4A). Of note, the nasopharyngeal carcinoma samples with normal EZH2 expression levels also showed a decreased expression of the genes associated with cell proliferation. Cellular proliferation was then examined in nasopharyngeal carcinoma cell cultures in order to determine whether EZH2 influences the proliferation of nasopharyngeal carcinoma cells. Let-7a induction and EZH2 knockdown by siRNA significantly reduced cellular proliferation in CNE-2 cells (Fig. 4B). Furthermore, the let-7a-treated cells demonstrated a significant increase in the number of cells in the G0/G1 phase in comparison to the untreated CNE-2 cells (Fig. 4C).

To determine the EZH2-mediated effects of let-7a on nasopharyngeal carcinoma cell apoptosis, we analyzed which genes associated with apoptosis correlated with EZH2 expression in the nasopharyngeal carcinoma and normal tissues. A significant correlation between the expression of 35 out of 424 genes associated with cell apoptosis and EZH2 expression was observed (Fig. 5A). In order to determine whether the let-7a upregulation or EZH2 inhibition also affected nasopharyngeal carcinoma cell apoptosis, Annexin V and PI double-staining was performed. The upregulation of let-7a resulted in a significant increase of apoptosis in the CNE-2 cells (Fig. 5B).

Having demonstrated EZH2 as a direct target of let-7a, we then examined the importance of EZH2 in let-7a-mediated cell proliferation and apoptosis. We first transfected EZH2 without 3’UTR into the CNE-2 cells. Western blot analysis showed that the transfection with EZH2 without 3’UTR overrided EZH2 expression targeted by let-7a (Fig. 6A). As shown in Fig. 6B, when we transfected the cells with EZH2 without 3’UTR and let-7a, the expression of EZH2 significantly abrogated the effect of let-7a on cell cycle distribution and apoptosis (Fig. 6B and C). These findings suggest that EZH2 is a major target of miR-106b involved in nasopharyngeal carcinoma cell proliferation and apoptosis.