| BackgroundNasopharyngeal carcinoma (NPC) has a remarkably unusual ethnic and geographic distribution in Southern China and Southeast Asia, especially those of Cantonese origin. Three major etiological factors of NPC include genetic susceptibility, environmental factors and Epstein-Barr virus (EBV) infection, but the molecular mechanism of its pathogenesis is not yet fully understood. Previous studies found that NPC had a family history, which suggested that genetic susceptibility might play an important role in the pathogenesis of NPC. Several reports demonstrated that the consistent non-random loss of heterozygosity (LOH) on the short arm of chromosome 3 (3p) was detected in NPC, with a especially high frequency of LOH on 3p14～21,3p21.3-22 and 3p25-26. Additionally, these genetic changes of 3p have taken place before EBV infection and malignant transformation of nasopharyngeal epithelial cells. Therefore, the potential tumor suppressors located in 3p might contribute to the development of NPC.MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs of 19～25 nucleotides in length that modulate gene expression post-transcriptionally. miRNAs are mostly transcribed from a hairpin intermediate of about 70～100 nucleotides called pre-miRNA, and undergo further processing by the ribonucleases Dicer. The mature miRNA targets the 3’untranslated region (3’UTR) of its target mRNA, and induces mRNA degradation. When miRNA and its target mRNA sequence show imperfect complementarities, translation into a protein is blocked. Most microRNA genes are found in intergenic regions or in anti-sense orientation to genes and contain their own miRNA gene promoter and regulatory units. As miRNAs involve in biological development, cell proliferation, differentiation and apoptosis, dysregulation of miRNAs appears to play a crucial role in cancer pathogenesis.Tumorigenesis is a complicated process which depends largely on the interaction of different genes. Besides protein-coding genes, miRNAs are regarded as a new player in cancer. It has been revealed that the deregulation of miRNAs is associated with a variety of cancers, and involved in the initiation and progression of cancer cells. Recent evidence has shown that about half of the human miRNAs are located in cancer-associated genomic regions, including minimal regions of LOH, minimal regions of amplification and fragile sites. Their abnormal expression plays a key role in the hallmarks of cancer, such as cell proliferation, differentiation, apoptosis, migration, invasion, metastasis and angiogenesis. These miRNAs have been confirmed to downregulate the expression of many cancer-related genes and function as oncogenes and tumor suppressor genes.MiR-26a was first cloned from Hela cells, with 21nt, and its expression had no tissue specificity. Notably, miR-26a is located in 3p22, which are fragile chromosomal regions with high frequency of LOH in NPC. The genomic location of 3p22 attracted our attention, and we were interested in the possible role of miR-26a in NPC. Recent studies have demonstrated that the abnormal expression of miR-26a is involved in innate immune response, cell differentiation, initiation and progression of multiple solid tumors and hematopoietic tumors. However, the role of miR-26a in cancer cells seemed controversial, as it was downregulated and played as a tumor suppressor in lymphoma, liver and breast cancer, but it was amplified and promote gliomagenesis in glioblastoma and glioma. Up to now, no functional evidence of miR-26a in NPC has been documented. The expression level of miR-26a and its possible role in NPC should be fully addressed.Based on the above findings, the aim of this study is to examine the expression of miR-26a, identify its possible role in NPC pathogenesis, and elucidate the molecular mechanisms of its suppressive or oncogenic activities on NPC. We hope that this study will improve the better understanding of NPC pathogenesis and the development of novel effective therapies for NPC.Materials and Methods1. Cell cultureThe human NPC cell lines 5-8F,6-10B, CNE1, CNE2, C666-1, HONE1, and HNE-1 were cultured in RPMI-1640 with 10% FBS and 1% Penicillin-Streptomycin antibiotic solution. An immortalized nasopharyngeal epithelial cell NP69 was cultured in Keratinocyte-SFM supplemented with bovine pituitary extract. HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM). All cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37℃.2. Clinical specimensPrimary NPC biopsy specimens and normal biopsies of thenasopharynx were obtained from Nanfang Hospital (Southern Medical University, Guangzhou, China). Both tumor and normal tissues were histologically confirmed by H&E (hematoxylin and eosin) staining. All NPC specimens were classified as undifferentiated nonkeratinizing type. Informed consent was obtained from each patient, and the research protocols were approved by the Ethics Committee of Nanfang Hospital.3. Vector construction(1) pLVTHM/miR-26a:This recombinant lentiviral vector can stably overexpress miR-26a, which was constructed by myself in Cancer Research Institute.(2) pGL3-wt EZH2 3’UTR and pGL3-mut EZH2 3’UTR:A 264-bp fragment of EZH2 3’UTR was amplified by PCR and cloned downstream of the firefly luciferase gene in pGL3 vector. The vector was named wild-type (wt) 3’UTR. Site-directed mutagenesis of the miR-26a binding site in EZH2 3’UTR was performed using GeneTailor Site-Directed Mutagenesis System and named mutant (mt) 3’UTR.(3) Lenti-EZH2 and Lenti-shEZH2:These two lentiviral vectors for cDNA and shRNA delivery of EZH2 were gifts of Dr. Chen from Chinese University of HongKong.4. Production and purification of lentivirusThe transfer vector and the packaging plasmids were co-transfected into 293T cells using CaPO4 precipitation. The supernatant was harvested 48-72h posttransfection. Then, the viral particles were concentrated by ultracentrifugation, and the viral pellets were resuspended. The titration of lentiviral suspension was calculated by making a tenfold serial dilution.5. Cell transfection(1) miRNA transfection:The transfection was performed using Lipofectamine 2000 reagent, and a working concentration of miRNA was 100nmol in a 6-well plate. (2) Lentivirus infection:The packaged lentivirus were used to infect NPC cell lines in 5 different MOIs (multiplicity of infection). At 48h transfection, the cells were observed using a fluorescence microscope to detect green fluorescent protein (GFP). Following fluorescence-activated cell sorting, the GFP-positive cells were isolated, and the cell lines were established, which could stably overexpress miR-26a, overexpress EZH2 and silence EZH2 respectively.(3) miRNA and plasmid co-transfection:The co-transfection was performed using Lipofectamine 2000 reagent, and a working concentration of miRNA was 50nmol, a working concentration of,pGL3-wt EZH2 3’UTR and pGL3-mut EZH2 3’UTR was 100ng, and a working concentration of an internal control pRL-SV40 vector was 5ng in a 24-well plate.6. Real-time PCRTotal RNA and small RNA were extracted from cells and tissues. The RNA was reversely transcribed into cDNA. Quantitative real-time PCR (qPCR) was performed using SYBR Green PCR master mix. All samples were normalized to internal controls and fold changes were calculated through relative quantification (2-△△Ct).7. Western blotTotal protein was isolated and quantitated using BCA assay. The protein lysates were separated by 10% SDS-PAGE, and electrophoretically transferred to PVDF membrane. Then, the membrane was incubated with antibodies and detected by chemiluminescence. The intensity of protein fragments was quantified with the Quantity One software.8. Luciferase reporter assayThe cells were seeded in 24-well plates and cotransfected with pGL3 constructs with or without miR-26a inhibitor or precursor for 24h. Each sample was also cotransfected with pRL-CMV plasmid expressing Renilla luciferase to monitor the transfection efficiency. At 24h post-transfection, the activity of firefly luciferase was measured by using the dual-luciferase reporter assay system as described by the manufacturer (Promega Corporation). Relative luciferase activity was normalized with Renilla luciferase activity and then compared with the pGL3 control.9. Cell biology experiments(1) MTT assay:A total of 1×103 cells were seeded in a 96-well plate and then allowed to grow in normal medium for 4 days. For MTT assay, cells were incubated in 20μl of 5mg/ml solution of MTT at 37℃for 4h and lysed in 150μl dimethyl sulfoxide (DMSO) at room temperature for 10min. The absorbance in each well was measured at 490 nm by a microplate reader.(2) Colony formation assay:The cells were plated in 6-well plates at 2×102 per well and grown for 2 weeks. After 2 weeks, the cells were washed twice with PBS, fixed with methanol/acetic acid (3:1, v/v), and stained with 0.5% crystal violet. The number of colonies was counted under the microscope.(3) Cell-cycle analysis:The cell-cycle distribution was analyzed by propidium iodide (PI) staining and flow cytometry.10. Animal experimentA total of 5×105 C666-1/con or C666-1/miR-26a cells were injected subcutaneously into the dorsal flank of nude mice. Each group contained 5 mice and the experiment was repeated 3 times. Tumor size was measured every 2 days. When subcutaneous tumors reached the volume of 500mm3, mice were sacrificed and tumors were dissected. Tumor volumes were calculated as follows: volume=(D×d2)/2, where D meant the longest diameter and d meant the shortest diameter11. ImmunohistochemistryFormalin-fixed, paraffin-embedded tissues of transplanted tumors were sectioned at 4-mm thickness and analyzed for Ki-67 and PCNA expression. Visualization was achieved using the EnVisionp peroxidase system. A sample was considered positive if more than 50% of the tumor cells retained nuclear staining, and 5 fields were randomly selected according to semiquantitative scales. The intensity of staining was scored manually (high,3; medium,2; low,1; no staining,0) by 2 independent experienced pathologists, and only tumor cells were scored.12. Statistical analysisSPSS 13.0 software was used for statistical analysis. Data were presented as Mean±SEM of at least 3 independent experiments. The results of MTT assay and tumor growth assay were analyzed by Factorial design analysis of variance. The relationship between EZH2 and miR-26a expression was explored by Spearman’s correlation. The result of immunohistochemistry was analyzed by Mann-Whitney U test. Two-tailed Student’s t test was used for comparisons of 2 independent groups. One-way ANOVA was used for comparisons of 3 or more than 3 independent groups, with the SNK (Student-Neuman-Keuls) tests for multiple comparisons. P values of< 0.05 were considered statistically significant.Results1. The expression level of miR-26a in NPC cells and tissuesA panal of human NPC cell lines (5-8F,6-10B, CNE1, CNE2, C666-1, HONE1 and HNE-1) was firstly analyzed to quantitate the expression level of miR-26a. The results of qRT-PCR showed that the expression of miR-26a was decreased in all seven NPC cell lines examined, compared with the immortalized nontumorigenic cell line NP69 (F=8.875, P<0.001). We further examined the expression of miR-26a in 18 NPC specimens and 16 normal nasopharyngeal epithelial tissues. Consistent with the data obtained from NPC cell lines, the average expression level of miR-26a was reduced by 60% in NPC specimens than in normal nasopharyngeal epithelial tissues (t=-6.976, P<0.001). All these results indicated that miR-26a was downregulated in NPC cells and tissues. We suggested that 3p LOH was the main reason for the abnormal expression of miR-26a in NPC.2. The growth-inhibitory effect of miR-26a on NPC cells in vitro and in vivoWe selected the undifferentiated C666-1 and low-differentiated HNE-1 cells, which excellently exhibited similar clinical phenotypes of NPC, as the most suitable model for further experiments. To explore the effect of miR-26a on cell growth, C666-1 and HNE-1 cells were transiently transfected with miR-26a mimic or miR-26a inhibitor, respectively. The results showed that the expression of miR-26a was upregulated by 4.99-fold (t=-9.650, P=0.001) in C666-1 and by 5.47-fold (t=-11.307, P<0.001) in HNE-1 cells. However, the expression of miR-26a was reduced by 85%(t=8.175, P=0.001) and 80%(t=8.143, P=0.001) in C666-1 and HNE-1 cells respectively after the transfection of antimiR-26a. All these data demonstrated that the transient transfection could effectively upregulate or downregulate the expression of miR-26a.The results of MTT assay displayed that miR-26a overexpression inhibited cell growth in C666-1 cells by 42%(t=21.682, P<0.001) and in HNE-1 cells by 45% (t=26.662, P<0.001) at 96h post-transfection, and miR-26a downregulation promoted cell growth by 55%(t=-40.993, P<0.001) and 47%(t=-28.592, P<0.001) respectively. The results of cell cycle distribution showed that C666-1 cells transfected with miR-26a displayed an increased percentage of cells in G1-phase (t=-15.128, P<0.001) and fewer cells in S phase (t=13.173, P<0.001). The cell cycle distribution did not have significant difference in C666-1 cells transfected with miR-26a inhibitor (P>0.05). These results suggested that the growth-suppressive effect of miR-26a was partly due to a G1-phase arrest.We next used lentiviral vectors to stably restore the expression of miR-26a in C666-1 and HNE-1 cells using different MOIs (multiplicity of infection), and examined cell growth rate and cell cycle distribution. We showed that the expression levels of miR-26a were increased by 9.46-fold (F=152.822, P<0.001) in C666-1 and 10.83-fold (F=152.871, P<0.001) HNE-1 cells respectively in a dose-dependent manner and reached a very high level at MOI 100. Therefore, the same condition (MOI=100) was applied for further experiments. Following fluorescence activated cell sorting, the GFP positive cells were isolated, and cell lines C666-1/miR-26a and HNE-1/miR-26a were established to stable overexpress miR-26a.The growth inhibition induced by LV-miR26a infection was similar to that induced by miR-26a mimic transfection, and a Gl-phase arrest was also observed in LV-miR26a-infected cells in a similar way (data not shown). As demonstrated in colony formation assay, the colony formation efficiency was reduced by 44% (t=25.154, P<0.001) in C666-1/miR-26a and 42%(t=22.321, P<0.001) in HNE-1/miR-26a cells respectively. We also performed tumor growth assay in vivo. The results showed that as ealy as nine days post implantation, the growth of transplanted tumors between C666-1/miR-26a and control cells became statistically significant (t=6.649, P<0.001). At two weeks after implantation, those mice injected with C666-1/miR-26a carried larger burdens. As compared with control, the average tumor volume of the LV-miR26a-treated group was markedly reduced by more than 45%(t=15.605, P<0.001). Additionally, both the staining intensity and the number of hyperproliferative Ki-67 positive and PCNA positive tumor cells were significantly decreased compared with control (P<0.05).All these results demonstrated that the abnormal expression of miR-26a is associated with cell proliferation and tumorigenicity of NPC cells, and it functions as a potential tumor suppressor in NPC pathogenesis.3. The molecular mechanisms of growth-inhibition induced by miR-26aWe used three database (miRBase, TargetScan and miRanda) to predict the target gene of miR-26a in NPC cells, and found that Enhancer of Zeste Homolog 2 (EZH2) was identified as a target gene. Based on the results of bioinformatics analysis, we suspected that EZH2 might be a target gene of miR-26a in NPC cell lines. To confirm this hypothesis, we constructed the recombinant plasmid pGL3-wt EZH2 3’UTR and mutant vector pGL3-mut EZH2 3’UTR, and performed dual luciferase reporter assay. The results showed a significant decrease of luciferase activity when miR-26a mimic and pGL3-wt EZH2 3’UTR vector were co-transfected in C666-1 cells (t=13.026, P<0.001), suggesting miR-26a could directly target the 3’UTR of EZH2 in NPC cells. In addition, a significant increase of luciferase activity was examined when antimiR-26a and pGL3-wt EZH2 3’UTR vector were co-transfected in C666-1 cells (t=-8.370, P=0.001).Next, we examined the expression of EZH2, when different MOIs were performed to overexpress miR-26a. The results indicated that ectopic expression of miR-26a led to a dose-dependent decrease of EZH2 mRNA and protein levels in C666-1 (F=88.649, P<0.001) and HNE-1 (F=442.510, P<0.001) cells respectively. Moreover, miR-26a silencing could upregulate EZH2 mRNA and protein levels (P<0.001). Lastly, we measured the mRNA levels of EZH2 in NPC specimens. The results demonstrated that the average expression level of EZH2 was upregulated by 2.12-fold in NPC specimens than in normal nasopharyngeal tissues (t=5.818, P<0.001). Moreover, when EZH2 mRNA levels were plotted against miR-26a expression, a significant inverse correlation was observed (r=-0.874, P<0.001). All these results suggested EZH2 was a target of miR-26a in NPC cells.To elucidate whether the growth-suppressive effect of miR-26a was mediated by repression of EZH2 in NPC cells, we performed gain-of-function and loss-of-function studies. We firstly silenced EZH2 to investigate whether the reduced expression of EZH2 could mimic the suppressive effect of miR-26a. The results of MTT assay showed that EZH2 knockdown reduced cell growth rate by 41% in C666-1 cells (F=612.085, P<0.001), similar to the suppressive effect of miR-26a. Cell cycle analysis also indicated that silence of EZH2 led to a G1-phase arrest (F=223.443, P<0.001). Subsequently, we evaluated whether ectopic expression of EZH2 could rescue the suppressive effect of miR-26a. C666-1/miR-26a cells were infected with LV-EZH2. We showed that ectopic expression of EZH2 significantly rescued miR-26a-induced cell growth inhibition (F=1078.465, P<0.001) and cell cycle arrest (F=360.732, P<0.001). These results suggested that the growth-inhibitory effect of miR-26a was mediated by repressing EZH2 in NPC cells.It is well-known that an average miRNA has approximately 100 target sites and regulates a large fraction of protein-coding genes, which form a regulatory network. To further explore the molecular mechanisms of growth-inhibition induced by miR-26a, we examined the expression of a panal of cell cycle regulators on p53 and pRb pathways. The p53 and pRb pathways are involved in the regulation of cell cycle progression and frequently deregulated in cancers. C-myc is one of the most important oncogenes which promote cell proliferation. We demonstrated that the levels of CCND3, CCNE2, CDK4, CDK6 and c-myc were decreased, while the levels of tumor suppressors p14ARF and p21CIP1 were increased after miR-26a overexpression and EZH2 silencing. However, their dysregulated expression did not affect the levels of CCND1, CCNE1, CDK2, p16IN4A, p53 and pRb. More importantly, we found that the deregulated expression of miR-26a effectors could be restored by overexpression of EZH2. In addition, we found that miR-26a overexpression could suppress the expression of CCND2 independent of EZH2, indicating that it was another target gene of miR-26a in NPC cells. All these results documented that miR-26a repressed EZH2 expression, which, in turn, by mediating G1-S checkpoint regulators, inhibited the growth and tumorigenicity of NPC cells.Conclusions1. The expression level of miR-26a is downregulated in NPC cells and tissues.2. The abnormal expression of miR-26a is associated with cell proliferation and tumorigenicity of NPC cells, which functions as a potential tumor suppressor in NPC pathogenesis.3. EZH2 is a target gene of miR-26a in NPC cells.4. MiR-26a represses EZH2 expression, which, in turn, by mediating G1-S checkpoint regulators, inhibits the growth and tumorigenicity of NPC cells. |
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