Abstract
Aim: To validate Epstein-Barr virus BamHI-A rightward transcript 7 microRNA (ebv-miR-BART7) expression in plasma from patients with nasopharyngeal carcinoma (NPC) and explore the oncogenic role of ebv-miR-BART7 in NPC cells. Patients and Methods: Plasma ebv-miR-BART7 levels were measured using real-time quantitative RT-PCR. Effects on cell proliferation, invasion, migration, and resistance to cisplatin were studied on NPC cells using real-time cell analyzer. Results: The plasma ebv-miR-BART7 level was significantly higher in patients with NPC in comparison with that from healthy individuals. The ebv-miR-BART7 was detectable in all the patient plasma samples and was independent of the EBV DNA level. In vitro expression of ebv-miR-BART7 enhanced proliferation, migration, and invasion of NPC cells. Furthermore, NPC cells expressing ebv-miR-BART7 were more resistant to cisplatin. High-throughput gene expression analysis suggested that ebv-miR-BART7 affects multiple cancer-related pathways. Conclusion: Our results indicate that plasma ebv-miR-BART7 could be used in NPC screening, especially in cases where EBV DNA is not detectable. The association of ebv-miR-BART7 with common oncogenic pathways suggests that ebv-miR-BART7 is a potential biomarker for undifferentiated NPC.
Nasopharyngeal carcinoma (NPC) is a common type of head and neck cancer and is related to Epstein-Barr virus (EBV) infection. The major histological subtype is undifferentiated carcinoma, with high incidence in Southern China and Southeast Asia (1). Undifferentiated NPC is closely associated with Epstein-Barr virus, the first human virus found to be related to the development of human malignancy. EBV infection is an early event and EBV frequently exists in the early stages in dysplastic lesions and carcinoma in situ (2). Based on the consistent association of NPC and EBV, the genetic materials of EBV have been used in the diagnosis and screening of patients with NPC.
MicroRNAs (miRNA) are small non-protein-coding RNAs which regulate mRNA at the post-transcriptional level. miRNAs are small (19-22 or 19-25 nucleotides) epigenetic regulators (3). miRNAs bind to their target mRNAs in a partial or complete complementary manner. They regulate gene expression by promoting target mRNA degradation and/or hindering mRNA translation (4). The EBV genome contains two major clusters, BamHI fragment H rightward open reading frame 1 (BHRF1) and BamHI-A rightward transcript (BART), coding for 25 precursors and 44 mature viral-encoded miRNA. ebv-miR-BART7 (MI0003729) is a viral-encoded miRNA, which is highly expressed in undifferentiated NPC cells latently infected with EBV. ebv-miR-BART7 was discovered in 2006, however, the extent of ebv-miR-BART7 expression in NPC was not explored at that time (5, 6). Mature ebv-miR-BART7 (MIMAT0003416) is a single-stranded molecule containing 22 nucleotides (5-caucauaguccaguguccaggg-3). A recent study using next-generation sequencing confirmed that ebv-miR-BART7 was highly expressed in NPC cells, suggesting that it might have an oncogenic role (7).
Here, we explored the use of circulating ebv-miR-BART7 in NPC screening and compared it with the circulating EBV DNA. Furthermore, we examined the role of ebv-miR-BART7 in the genesis of NPC cells and explored the potential functional pathways affected by ebv-miR-BART7.
Patients and Methods
Patient samples. Peripheral blood samples were obtained from patients with NPC at the Department of Surgery, The University of Hong Kong, Queen Mary Hospital, Hong Kong. Peripheral blood samples were collected from 41 patients with primary NPC, 21 with recurrent NPC and from 21 healthy volunteers. The patients with primary NPC included 34 males and seven females, their age ranging from 23-79 years. There were six patients with T1 stage disease, 24 with T2, 10 with T3 and one with T4. The patients with recurrent NPC included 17 males and four females, aged from 29-75 years; there were seven patients with T1 disease, three with T2, seven with T3, and four with T4.
Cell cultures. NPC cell lines HK1 (Hong Kong, China) and HONE1 (Hunan, China) were maintained in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco). HK1 is derived from well-differentiated squamous carcinoma (8); HONE1 is derived from poorly differentiated squamous carcinoma (9).
Immunocytochemistry. NPC cells were washed with PBS and fixed with 2% paraformaldehyde overnight. The nucleus was stained by blue-fluorescent 4’,6-Diamidino-2-Phenylindole (DAPI) (Invitrogen, Grand Island, NY, USA); F-actin was labeled in red with Alexa Fluor® 635 phalloidin (Invitrogen, Grand Island, NY, USA). The morphologies of HONE1 and HK1 transfected with 1.5 nM ebv-miR-BART7 (BART7) mimic or negative control siRNA were observed.
RNA extraction and real-time quantitative RT-PCR analysis. Total RNA was extracted with TRIZOL (Invitrogen) following the manufacturer's protocol. RNA was converted to cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, California, USA). Transcripts levels of ebv-miR-BART7 and U6 control snRNA were measured by TaqMan Gene Expression Assays (Applied Biosystems) using a LightCycler® 480 (Roche Applied Science, Indianapolis, IN, USA).
EBV DNA quantification. Absolute EBV DNA quantification was performed using LightMix® Kit for the detection of EBV (TIB MOLBIOL, Berlin, Germany). EBV DNA purified from 200 μl plasma by High Pure Viral Nucleic Acid Kit (Roche Applied Science) was amplified using a LightCycler® 480 (Roche Applied Science). Absolute EBV quantification was calculated using a standard curve derived from 101-106 copies of EBV DNA.
Proliferation, migration and invasion assay. Proliferation assay was performed on E-Plate 16 using a RTCA DP instrument (Roche Applied Science). Real-time migration and invasion monitoring was performed using CIM-Plate 16 on a RTCA DP instrument. HONE1 and HK1 transfected with 1.5 nM ebv-miR-BART7 mimic or negative control siRNA using Hiperfect transfection reagent (QIAGEN, Valencia, CA, USA) were seeded on E-Plate 16 or CIM-Plate 16. For invasion assay, the upper chamber of CIM-Plate 16 was coated with Matrigel (1:30 dilution; BD Biosciences, San Jose, California, USA) for 4 hours at 37°C. The culture medium containing 10% FBS was added to the lower chamber as a chemo-attractant.
Cisplatin-resistance assay. After transfection with 1.5 nM ebv-miR-BART7 mimic or negative control siRNA for 72 hours, HONE1 and HK1 were harvested and seeded on E-plate 16. After 26 hours, cisplatin was added to the medium at a final concentration of 17 μM, followed by real-time cell index monitoring for 23 hours. The cell index of NPC cells transfected with BART7 mimic was compared to that of NPC cells transfected with negative control siRNA.
Microarray analysis. The RNA quality assessment, gene expression microarray and data analysis were carried out at the Genome Research Centre of the University of Hong Kong. GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA) was used for global gene expression profiling. Microarray data analysis was performed with GeneSpring GX version 10.0 (Agilent Technologies, Santa Clara, CA, USA). Median normalization of log2-transformed data was used. The differentially expressed genes were evaluated by DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov/home.jsp).
Statistical analysis. Statistical analysis was performed with SPSS V16.0 (Armonk, New York, USA).
Results
Circulating ebv-miR-BART7 levels in patients with NPC and healthy individuals. Figure 1A shows the serological ebv-miR-BART7 levels in our cohort. Patients with primary NPC had significantly higher plasma ebv-miR-BART7 levels in comparison with the healthy individuals (p<0.001, Mann-Whitney U-test) (Figure 1A). Figure 1A also shows the receiver operating characteristic (ROC) analysis for circulating ebv-miR-BART7 in our cohort. The area under the curve (AUC) was 0.81 (Figure 1A).
Comparison of circulating EBV DNA and ebv-miR-BART7 in NPC patients. EBV DNA was detected in 18 out of 41 (44%) plasma samples of patients with NPC (Figure 1B). In healthy plasma, the EBV DNA level was below the detectable level in all 21 cases (Figure 1B). The plasma EBV DNA level was significantly higher in patients with primary NPC in comparison with the healthy controls (p=0.024, Mann-Whitney U-test) (Figure 1B). In comparison, ebv-miR-BART7 was detected in all 41 (100%) primary NPC cases and the ebv-miR-BART7 level was independent of the circulating EBV DNA level (Figure 1C). Furthermore, a high level of ebv-miR-BART7 was observed in both serologically EBV DNA-positive (p<0.001, Mann-Whitney U-test) and -negative (p=0.001, Mann-Whitney U-test) primary NPC cases in comparison with healthy controls (Figure 1C).
Circulating ebv-miR-BART7 and EBV DNA level in recurrent NPC patients. In comparison with primary NPC, patients with recurrent NPC had a lower serological ebv-miR-BART7 level (p<0.001, Mann-Whitney U-test) (Figure 1D). Although the median serological ebv-miR-BART7 level was higher in patients with recurrent NPC in comparison with the healthy controls, no statistically significant difference was observed. Both primary NPC and recurrent NPC had significantly higher plasma EBV DNA levels in comparison with the healthy controls (Figure 1E).
Effects of ebv-miR-BART7 on proliferation, migration, and invasion of NPC cells. To explore the functional role of ebv-miR-BART7 on NPC cells, ectopic expression of ebv-miR-BART7 was performed on 2 EBV-negative NPC cell lines HONE1 and HK1. Overexpression of ebv-miR-BART7 was observed in HONE1 and HK1 transfected with ebv-miR-BART7 mimic (Figure 2A). In the presence of ebv-miR-BART7, HONE1 demonstrated a significant increase in the rate of proliferation (Figure 2B), migration (Figure 2C) and invasion (Figure 2D). For HK1, in the presence of mature ebv-miR-BART7, we only observed an increase in migration (Figure 2C) and invasion (Figure 2D). ebv-miR-BART7 expression had no significant effects on the proliferation rate of HK1 cells (Figure 2B).
Cisplatin sensitivity of ebv-miR-BART7-expressing NPC cells. To examine the role of ebv-miR-BART7 in response to cisplatin treatment, we transfected the NPC cell lines HONE1 and HK1 with ebv-miR-BART7 mimic and exposed the cells to cisplatin. Figure 3 shows that ebv-miR-BART7-expressing cells were more resistant to cisplatin in comparison with the mock control. Both HONE1 and HK1 cells demonstrated significant resistance to cisplatin in the presence of ebv-miR-BART7.
Functional pathways affected by ebv-miR-BART7 in the NPC HONE1 cells. We examined the global gene expression alterations in the undifferentiated NPC cells HONE1 induced by ebv-miR-BART7 with microarray analysis. With a fold-change cut-off of 3 and p<0.05, 625 genes were found to be differentially expressed in the presence of ebv-miR-BART7 (Table I). Pathway analysis indicated that ebv-miR-BART7 expression affects the signaling pathway of calcium (p=0.003) and of the immune system (p=0.013), ionotropic glutamate receptor (p=0.016), ATP-binding cassette (ABC) transporters (p=0.019), nuclear receptors in lipid metabolism and toxicity (p=0.029), transforming growth factor (TGF)-signaling pathway (p=0.035), dilated cardiomyopathy (p=0.037,) opioid signaling (p=0.04), and metabolism of lipids and lipoproteins (p=0.045) (Table II).
Discussion
EBV DNA is now regarded as a molecular marker for NPC diagnosis and screening. The major drawback is that EBV DNA is not present in all patients with NPC. Regional data suggested that a subgroup of patients with NPC is serological negative for circulating EBV DNA (10). Yuan et al. demonstrated that EBV DNA is present only in 67% of NPC cases (11). In a study using mixed plasma and serum samples, 41% (69/176) of patients with NPC were serologically negative for EBV DNA (12). In low-risk areas, such as Europe, EBV DNA is also not an absolute marker. Patients with EBV-encoded small RNA (EBER)-negative NPC had no detectable EBV DNA in their circulation. Thus, in cases where both EBV antibody and EBV DNA are lacking, there will be no suitable marker available for NPC detection/monitoring.
As shown in our results, both patients with NPC and healthy individuals had detectable circulating ebv-miR-BART7 in the plasma. Similar to EBV DNA, ebv-miR-BART7 was significantly higher in patients with NPC in comparison with the healthy controls. To test our hypothesis that ebv-miR-BART7 could be used as a serological marker in cases where no circulating EBV DNA is found, we analyzed the plasma ebv-miR-BART7 levels according to the detection rate of EBV DNA in the plasma samples. High plasma ebv-miR-BART7 was detected in patients with NPC and the circulating level is independent of the level of circulating EBV DNA. Intra-categorical analysis revealed that ebv-miR-BART7 levels are similar in patients with NPC regardless of serological EBV DNA detection. Furthermore, ROC analysis indicated that serological ebv-miR-BART7 is useful to differentiate patients with NPC from healthy individuals (p=0.0002).
To examine the functional role of ebv-miR-BART7 in NPC, we transfected the ebv-miR-BART7 mimic into the NPC-derived cell lines and examined the behavioral changes. HONE1 was originally derived from NPC tissue which was EBV positive. Prolonged culture and continuous passaging resulted in the loss of EBV genome in NPC HONE1 cell. In contrast, HK1 is derived from differentiated NPC which was originally EBV negative. Interestingly, ebv-miR-BART7 had a positive effect on all the examined cancer cell properties, especially in the undifferentiated NPC cell line HONE1. ebv-miR-BART7 increased cell proliferation, promoted cell migration and increased invasion. In addition, in the presence of ebv-miR-BART7, both NPC cell lines became more resistant to cisplatin, a common chemotherapeutic agent used in NPC treatment. The results indicated that ebv-miR-BART7 plays a critical functional role and has an augmentative impact on the aggressive phenotype in NPC.
The gene products of EBV can alter somatic gene expression by controlling the biogenesis machinery of the host cells (13). We therefore suggested that the ebv-miR-BART7 would also have the same effect and control the transcriptome at post-transcriptional levels. In view of the fact that ebv-miR-BART7 induced multiple behavioral changes in NPC cells, we tried to identify the relevant biological pathways with microarray analysis and performed functional grouping of the differentially expressed genes using DAVID. Pathway analysis revealed that ebv-miR-BART7 alters multiple cancer-related pathways in undifferentiated NPC cells, implying that ebv-miR-BART7 is a critical player in the pathogenesis of undifferentiated NPC.
In conclusion, our results confirmed that ebv-miR-BART7 is highly expressed in NPC and the result is concordant with other similar studies. ebv-miR-BART7 is a critical viral oncomir as it can affect a myriad of functional pathways of NPC cells. By identifying suitable cut-off levels in further and larger sample sets, ebv-miR-BART7 might possibly be used in serological monitoring of patients with NPC, especially in cases where serological EBV DNA is not applicable. Further studies are warranted to explore the clinical use of EBV-derived miRNA in diagnosis, prognosis and follow-up management of patients with undifferentiated NPC.
Acknowledgements
This study was supported by Seed Funding of Basic Research, The University of Hong Kong, Li Shu Pui Professorship, The University of Hong Kong, Hong Kong Area of Excellent Scheme and Hong Kong UGC.
Footnotes
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↵* These Authors contributed equally to this study.
- Received April 22, 2012.
- Revision received June 27, 2012.
- Accepted June 29, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved