Circulating Epstein-Barr virus microRNA profile reveals novel biomarker for nasopharyngeal carcinoma diagnosis

Abstract

Nasopharyngeal carcinoma (NPC), a tumor quite prevalent in Asia, is closely associated with Epstein-Barr virus (EBV) infection status. Many NPC patients are not able to be treated in time when being diagnosed at an advanced stage. EBV-encoded microRNAs are reliable sources of biomarkers for NPC diagnosis. In this study, we conducted circulating EBV microRNAs profiling by quantitative reverse transcription polymerase chain reaction (qRT-PCR) among plasma samples of 159 NPC patients versus 145 normal controls (NCs) and serum samples of 60 NPC patients versus 60 NCs. Among the 44 mature EBV-encoded miRNAs, only miR-BART19-3p in plasma was proved to be significantly up-regulated in NPC patients ( $P<$ 0.05; fold change (FC) $>$ 2.0). The area under the receiver operating characteristic curve (AUC) for the signature to discriminate NPC patients from NCs was 0.848 with the sensitivity and specificity being 71.7% and 72.3%, respectively. The identified biomarker was analyzed in tissue specimens (44 NPC VS. 32 NCs) and proved to be consistently up-regulated in NPC tumor tissues. Bioinformatics analysis was further conducted to predict the potential targets of miR-BART-19-3p, which provided some hints to its close relationship with NPC development. In conclusion, we identified a novel biomarker – plasma miR-BART19-3p for the detection of NPC.

Keywords

Nasopharyngeal carcinoma Epstein-Barr virus circulating microRNAs biomarker diagnosis

1. Introduction

Nasopharyngeal carcinoma (NPC) is a cancer rising from nasopharynx epithelium [1]. Unlike other head and neck cancers, NPC has remarkably skewed ethnic and geographic distributions [2]. In certain regions such as southeast Asia and northern Africa, NPC has relatively higher prevalence than elsewhere, with viral infection, genomic landscape and some environment factors implicated in its causation [3, 4, 5, 6, 7]. Though the incidence and mortality rate of NPC has undergone gradual decrease recently due to advances in disease screening and control strategies, it is still a non-negligible health threat to numerous patients especially those in current Asia where the improvement of NPC prevention and control is still in urgent need [3, 8, 9, 10].

Epstein-Barr virus (EBV), the first human carcinogenic virus to be discovered, has been proved to be consistently associated with NPC, especially the undifferentiated type which makes up the majority in high risk areas [11, 12, 13]. Many EBV-encoded genes that are expressed in infected NPC cells (LMP1, LMP2, EBNA1, viral noncoding RNAs, etc.) play important roles in NPC pathogenesis [14]. Latent membrane protein 1 (LMP1), which has been frequently studied, is considered to be the first viral protein associated with cell malignant transformation [15].Besides, transcripts from the BamHI-A region (BARTs) and BamHI H rightward reading frame 1 region (BHRF1) are termed as the templates for the precursors of 44 mature microRNAs (miRNAs) [16].

MiRNAs are family of small non-coding RNAs (typically 18–25 nucleotides long) functioning in post-transcriptional gene expression regulation [17, 18]. Emerging evidence has revealed the tight involvement of miRNAs in tumor-correlated biological processes such as apoptosis, proliferation, and invasion in various cancer types [19]. EBV-encoded miRNAs are expressed through all infection phases and can influence a variety of cellular processes by regulating a broad range of viral and host genes [16, 20, 21]. According to previous studies, EBV miRNAs play important roles in the development of some hematological malignancies including EBV-induced lymphomagenesis, by manipulating several cancer-related mechanisms such as immune escape, inhibition of apoptosis, promotion of invasion and metastasis [22]. For NPC, quite a number of studies have found the over-expression of EBV-encoded miRNAs in tumor tissues, which are closely linked to immune regulation, tumorigenesis and progression by targeting specific genes such as PTEN, IPO7, and PUMA [23, 24, 25, 26, 27]. Moreover, aberrant expression of circulating EBV miRNAs among NPC patients, such as up-regulated miR-BART7 and miR-BART13 in plasma, further shows the potential of circulating EBV-encoded miRNAs as novel biomarkers for NPC detection [28]. In fact, stable existence of extracellular miRNAs in blood circulation has long established a good foundation for the discovery of reliable tumor biomarkers [19, 29]. Up to now, several human miRNAs in serum or plasma such as miR-22, miR-572, miR-638, miR-1234, miR-17, and miR-20a have been proposed as potential diagnostic signatures for NPC [30, 31]. However, studies focused on circulating EBV-encoded miRNAs and their value for NPC diagnosis are still insufficient and limited.

Though several NPC biomarkers such as plasma EBV-DNA have been well-studied, quite a surprising proportion of NPC cases are diagnosed at an advanced stage which have poor response to conventional treatment [32, 33]. In present study, we conducted comprehensive analysis of all the 44 mature EBV-encoded miRNAs in plasma and serum, with the hope of identifying novel signatures for NPC detection. Expression levels of the identified miRNAs were further explored in tissue samples. Should high-promising EBV miRNA biomarkers being discovered, it may benefit minimally invasive detection of NPC in the future.

2. Materials and methods

2.1 Participants and characteristics of study cohort

A total of 219 histopathologically confirmed NPC patients and 205 healthy donors (normal controls (NCs)) from First Affiliated Hospital of Nanjing Medical University and Jiangsu Cancer Hospital were enrolled in our study during 2016 to 2017.Their clinical and demographics characteristics were given in Table 1. There was no significant difference in gender and age distribution between NPC patients and NCs ( $P>$ 0.05). Written informed consent was obtained from each participant. The study was conducted after the approval of the Institutional Review Board of First Affiliated Hospital of Nanjing Medical University and Jiangsu Cancer Hospital (ID: 2016-SRFA-148; 2016-SRFA-149).

2.2 Study design

The list of 44 mature EBV-encoded miRNAs was obtained from the miRBase database (http://www. mirbase.org, Supplementary Table S1). Expression levels of the 44 miRNAs were preliminarily analyzed by qRT-PCR among plasma samples from 64 NPC patients and 64 NCs, and serum samples from 60 NPC patients and 60 NCs. The identified differentially expressed miRNAs were then confirmed among larger study cohorts. Moreover, expression analysis of the identified miRNAs was also conducted in 44 NPC tumor tissue specimens and 32 normal nasal mucosa tissue specimens. The flow chart of experiment design was shown in Fig. 1.

Figure 1.

The flow chart of experiment design. (NPC: nasopharyngeal carcinoma; NC: normal control; qRT-PCR: quantitative reverse transcription polymerase chain reaction.)

2.3 Sample preparation

All the participants were untreated when venous blood samples were drawn. Ethylene diamine tetra acetic acid (EDTA)-containing tubes (Becton, Dickinson and Company) or SST Advance tubes (Becton, Dickinson and Company) were used to collect whole blood samples for the separation of plasma or serum within 12 hours. The centrifugal process was 350 RCF (reactive centrifugal force) for 10 min followed by 20,000 RCF for 10 min for plasma isolation, and 1,500 RCF for 10 min followed by 12,000 RCF for 2 min for serum isolation. The obtained cell-free plasma and serum samples were stored at $-$ 80 ${}^{\circ}$ C for future analysis. Ultimately, a total of 304 plasma samples (159 NPC VS. 145 NCs) and 120 serum samples (60 NPC VS. 60 NCs) were applied for circulating miRNA expression analysis in this study. In addition, frozen tumor tissue specimens from NPC patients undergoing surgical operation and normal paraffin-embedded nasal mucosa tissue specimens from healthy donors (44 NPC VS. 32 NCs) were also collected and stored in liquid nitrogen until use.

Table 1
Demographic and Clinical characteristics of NPC patients and NCs

	Plasma		Serum		Tissue
Variables	Cases (%)	Control (%)	Cases (%)	Control (%)	Cases (%)	Control (%)
Number	159	145	60	60	44	32
Gender
Man	121 (76.1)	110 (75.9)	47 (78.3)	44 (73.3)	29 (65.9)	20 (62.5)
Woman	38 (23.9)	35 (24.1)	13 (21.7)	16 (26.7)	15 (34.1)	12 (37.5)
Age
$<$ 60	112 (70.4)	100 (69.0)	43 (71.7)	41 (68.3)	32 (72.7)	27 (84.4)
$\geqslant$ 60	47 (29.6)	45 (31.0)	17 (28.3)	19 (31.7)	12 (27.3)	5 (15.6)
TNM stage
I	2 (1.3)		1 (1.7)		26 (59.1)
II	32 (20.1)		11 (18.3)		13 (29.6)
III	79 (49.7)		27 (45.0)		3 (6.8)
IV	46 (28.9)		21 (35.0)		2 (4.5)
EBV DNA copy number
$ï ¼ œ$ 5.00E+02	68 (42.8)		25 (41.6)		28 (63.7)
$\geqslant$ 5.00E+02	87 (54.7)		34 (56.7)		16 (36.3)
NA	4 (2.5)		1 (1.7)
Pathological type
PDSCC	116 (73.0)		44 (73.3)		41 (93.2)
Non-keratinizing carcinoma	13 (8.1)		5 (8.3)		1 (2.3)
NA	30 (18.9)		11 (18.4)		2 (4.5)

NPC: nasopharyngeal carcinoma; NCs: normal controls; EBV: Epstein-Barr Virus; PDSCC: poorly differentiated squamous cell carcinoma; NA: not available; TNM stage: tumor/node/metastasis stage (refers to the 8th edition of the American Joint Committee on Cancer (AJCC) TNM system).

2.4 RNA extraction

Total RNA was extracted from 200 $\mu$ l plasma or serum using the mirVana PARIS Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instruction. After the addition of denaturing solution (Ambion, Austin, TX, USA), 5 $\mu$ l of synthetic C.elegans miR-39 (5 nM/L, RiboBio, Guangzhou, China) was added to each sample to normalize sample-to-sample variation. For tissue specimen, Trizol (Invitrogen, Carlsbad, CA, USA) was applied for RNA extraction. Total RNA was lysed into 100 $\mu$ l RNase-free water and stored at $-$ 80 ${}^{\circ}$ C for future analysis. RNA concentration and purity was measured using the Nanodrop 2000 thermo scientific spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

2.5 Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

In accordance with our previous studies [34], Bulge-Loop ${}^{\rm TM}$ miRNA qRT-PCR Primer Set (RiboBio, Guangzhou, China) with specific primers of reverse transcription (RT) and polymerase chain reaction (PCR) and was applied to amplify miRNAs. The RT reaction was performed following the procedure of 42 ${}^{\circ}$ C for 60 min and then 70 ${}^{\circ}$ C for 10 min. The PCR reaction was performed in the condition of 95 ${}^{\circ}$ C for 20 sec, followed by 40 cycles of 95 ${}^{\circ}$ C for 10 sec, 60 ${}^{\circ}$ C for 20 sec and then 70 ${}^{\circ}$ C for 10 sec. The reactions were conducted on the 7900HT real-time PCR system (Applied Biosystems, Foster City, CA, USA). SYBR Green Dye (SYBR ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ Premix Ex TaqTM II, TaKaRa, Dalian, China) was used to determine the relative amount of PCR products by fluorescence level. MiRNA expression level relative to cel-miR-39 and RNU6B ( $U6$ , for tissue) as was calculated using the 2 ${}^{-\Delta\Delta\text{Ct}}$ method [35]. In this study, no suitable reference gene had been discovered for the normalization of EBV-encoded miRNAs in blood circulation.

2.6 Statistical analysis

The expression levels of plasma and serum miRNAs in NPC patients and NCs were compared using Mann-Whitney U test. One-way ANOVA or $\chi^{2}$ test was used to evaluate the balance of demographic and clinical characteristic distribution among different study cohorts and measure the confounding effects of several clinical factors. The receiver operating characteristic (ROC) curve analysis was conducted and the area under the ROC curve (AUC) was measured to confirm the diagnostic value of identified miRNA biomarkers. Cox’s proportional hazards model was used to evaluate potential prognostic factors related to overall survival (OS) rate of NPC patients. All the data analysis and graph drawing was carried out using SPSS 22.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7.0 (GraphPad Software, USA). A two-sided $P<$ 0.05 was set to be of statistical significance.

3. Results

3.1 EBV miRNA expression profiling in plasma and serum samples

As shown in Fig. 1, we conducted primary overall expression analysis by qRT-PCR among plasma samples from 64 NPC patients versus 64 NCs, and serum samples from 60 NPC patients versus 60 NCs, to identify candidate EBV miRNAs as biomarkers for NPC diagnosis. MiRNAs with mean Ct-value more than 30 were excluded from our candidate lists to ensure the considerable concentration of marker miRNAs Among the 5 EBV miRNAs with mean Ct-value $<$ 30 (miR-BHRF1-2-5p, miR-BART7-3p, miR-BART18-5p, miR-BART12 and miR-BART19-3p), only miR-BART19-3p in plasma (64 NPC VS. 64 NCs) showed significant expressing difference with $P<$ 0.05 and FC $>$ 2.0 (Table 2). The identified miRNA was further analyzed among a larger cohort of plasma samples (95 NPC VS. 81 NCs), and the statistical trend of upregulation in NPC patients remained stable. In all, we detected a total of 159 NPC and 145 NCs plasma samples to evaluate the expression level of miR-BART19-3p, increasing trend could be observed in NPC patients in comparison to NCs (Fig. 2). We further compared the expression levels of plasma miR-BART19-3p between 64 NPC patients and 95 NPC patients in the two separate stages, and no significant difference was discovered ( $P>$ 0.05). For serum samples, none of the 5 miRNAs were found to be differentially expressed between the case and control groups (60 NPC VS. 60 NCs; $P>$ 0.05; Table 2). Therefore, further research of EBV miRNA expression in serum was stopped in this study.

Table 2
Expression levels of miRNAs (mean Ct-value $<$ 30) in serum and plasma samples. (presented as mean $\pm$ SD; $\Delta$ Ct, relative to cel-miR-39)

Clusters	Mature miRNAs	Plasma				Serum
		NPC ( $n=$ 64)	NC ( $n=$ 64)	FC	$P$ -value	NPC ( $n=$ 60)	NC ( $n=$ 60)	FC	$P$ -value
BHRF1-cluster	ebv-miR-BHRF1-2-5p	1.40 $\pm$ 1.89	1.77 $\pm$ 2.00	1.30	0.084	3.24 $\pm$ 2.02	3.58 $\pm$ 1.64	1.26	0.831
	ebv-miR-BART-7-3p	2.92 $\pm$ 1.53	2.85 $\pm$ 1.84	0.96	0.854	2.84 $\pm$ 2.49	3.05 $\pm$ 1.73	1.16	0.512
BART-cluster2	ebv-miR-BART-18-5p	0.07 $\pm$ 2.94	0.07 $\pm$ 3.00	1.00	0.521	$-$ 1.04 $\pm$ 4.32	$-$ 0.69 $\pm$ 4.20	1.27	0.508
	ebv-miR-BART-12	3.68 $\pm$ 4.43	3.98 $\pm$ 4.89	1.23	0.260	2.38 $\pm$ 2.53	2.81 $\pm$ 2.50	1.35	0.461
	ebv-miR-BART-19-3p	3.06 $\pm$ 1.52	5.22 $\pm$ 1.34	4.48	$<$ 0.001	5.51 $\pm$ 3.16	5.58 $\pm$ 3.33	1.05	0.886

NPC: nasopharyngeal carcinoma; NCs: normal controls; FC: fold change.

Figure 2.

Expression levels of the identified miRNA (ebv-miR- BART19-3p in plasma) among a total of 159 NPC patients and 145 normal controls. (N: normal control; T: tumor; Horizontal line: mean with 95% CI; **** $P$ -value $<$ 0.0001.)

Taken together, after overall exploration in both plasma and serum, we screened out only one potential biomarker from the 44 mature EBV miRNAs – plasma miR-BART19-3p for the diagnosis of NPC patients.

3.2 Diagnostic value of the identified EBV miRNA for NPC

ROC curve analysis was conducted and the AUC was calculated to evaluate the diagnostic performance of plasma miR-BART19-3p for NPC detection. As can be seen in Fig. 3, the identified EBV miRNA biomarker performed quite well in discriminating NPC patients from NCs with the corresponding AUC being as high as 0.848 (95% CI: 0.807–0.889; sensitivity $=$ 71.7%, specificity $=$ 72.3%; 159 NPC VS. 145 NCs). Figure 4 demonstrated the diagnostic capability of the identified miRNA for NPC patients in different stages. It was concluded that plasma miR-BART19-3p had good diagnostic efficacy in both early and late patients, with AUCs being 0.749 (95% CI: 0.639–0.858; Fig. 4A) and 0.819 (95% CI: 0.765–0.872; Fig. 4B), respectively.

Figure 3.

ROC curve analysis of ebv-miR-BART19-3p for NPC detection. (159 NPC VS. 145 NCs; ROC curve: receiver-operating characteristic curve; AUC: area under the ROC curve.)

Figure 4.

ROC curve analysis of plasma ebv-miR-BART19-3p for the detection of NPC patients in different stages in comparison with NCs. A: Stage I $+$ II VS. NCs; B: Stage II $I$ +IV VS. NCs. (ROC curve: receiver-operating characteristic curve; AUC: area under the ROC curve.)

3.3 Prognostic value assessment of the identified EBV miRNA signature

To determine whether plasma miR-BART19-3p was of prognostic value for NPC patients or not, we further constructed Cox proportional hazards model and conducted Kaplan-Meier curve analysis. Potential confounding factors including gender, age, TNM classification, tumor stage, lymph node status, and plasma EBV-DNA copy number were also measured. According to multivariate Cox regression analysis, no clear correlation was found between plasma miR-BART19-3p expression level and the prognostic conditions of NPC patients ( $P>$ 0.05, Supplementary Table S2). Alternatively, patients with later lymph node stage might have worse prognostic outcomes ( $P<$ 0.05). The conclusion of Kaplan-Meier curve analysis remained the same (data not shown).

3.4 Subgroup analysis for various clinicopathologic features

In order to avoid potential confounding effects of several clinicopathologic features and reveal their probable relationship with plasma miR-BART19-3p expression level, we further conducted subgroup analysis among 159 NPC patients and 145 NCs. For each of the analyzed factors including age, gender, tumor stage, pathological classification, and plasma EBV-DNA level, the expression levels of plasma miR-BART19-3p turned out to be consistent between different subgroups and all exhibited distinctive difference compared to NCs (data not shown).

3.5 Expression level of the identified EBV miRNA in tissue specimens

Expression level of miR-BART19-3p was further detected in tissue specimens (44 NPC VS. 32 NCs) to decipher its potential origin. As can be seen in Fig. 5, miR-BART19-3p was significantly up-regulated in NPC tumor tissues than in normal tissues ( $P<$ 0.05). Over-expression of miR-BART19-3p was observed both in plasma and tumor tissue samples.

Figure 5.

Expression levels of ebv-miR-BART19-3p in frozen tumor tissue specimens from 44 NPC patients and normal paraffin-embedded nasal mucosa tissue specimens from 32 healthy donors. (N: normal control; T: tumor; Horizontal line: mean with SEM; **** $P$ -value $<$ 0.0001.)

3.6 In silico target prediction of ebv-miR-BART19-3p

The seed sequence of ebv-miR-BART19-3p obtained from miRBase was input into TargetScanHuman Custom 5.2 (http://www.targetscan.org/vert_50/seedmatch.html) to predict its potential regulatory tar-gets [36]. A total of 992 genes were predicted to be targeted by ebv-miR-BART19-3p. The target gene list was input into the Functional Annotation Tool of DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/) to identify pathways probably regulated by ebv-miR-BART19-3p [37, 38]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed 51 potentially involved pathways with the modified Fisher $P$ value $<$ 0.05. Numerous cancer-associated pathways such as Proteoglycans in cancer, Wnt signaling pathway, FoxO signaling pathway and VEGF signaling pathway showed possibility to be dysregulated by miR-BART19-3p. Notably, in this list, Pathways in cancer of KEGG database were involved with 38 potential target genes, including oncogenes such as CBL and KRAS, tumor suppressor genes such as APC and DCC, cell cyclin-related genes such as CDC42 and CDK2, apoptosis-related genes such as PTGER2 and others, which revealed the close link of miR-BART19-3p to tumor biological processes. Viral carcinogenesis (20 genes) which included EBV infection-correlated NPC was listed out, which might reveal the role of EBV-encoded miR-BART19-3p in tumorigenesis. Moreover, Gene Ontology (GO) biological process analysis also showed various tumor associated biological processes that might be targeted by miR-BART19-3p ( $P<$ 0.05), which deserved precise verification in the future. The results were listed in Supplementary Table S3.

4. Discussion

Thanks to the rapid development of monitoring and treatment strategies for NPC, the overall incidence and mortality rate of NPC has undergone gradually decline during the last decades [3, 9, 10]. Besides several routine screening methods, novel molecular biomarkers such as circulating cell-free EBV DNA load, aberrant methylation of CDH13 gene from nasopharyngeal swabs, and elevated antibody titers against EBV components have continuously been discovered and developed as auxiliary diagnostic or prognostic predictors for NPC [39, 40, 41, 42]. miRNA, with its stable existence in blood circulation, has also been proved as a promising source of tumor biomarker [43, 44]. Several previous studies have reported some dysregulated circulating miRNAs of human origins for NPC detection [31, 45, 46]. However, unlike other cancers with unapparent causes, a majority of NPC cases are highly involved with the infection of EBV, a well-studied carcinogenic virus that is able to regulate certain miRNA expression [43, 47]. Apart from human miRNAs, aberrant expression of EBV-encoded miRNAs has also been frequently observed in both tumor tissues and blood circulation in NPC [28, 48, 49]. Actually, according to a high-throughput research carried out by Wong et al., miRNAs encoded by EBV were generally more up-regulated in NPC than those of human origins, and the expression levels of some circulating EBV miRNAs had distinctively positive correlation with the corresponding cellular amounts, which further demonstrated the great potential of EBV-encoded miRNAs as reliable biomarkers for NPC diagnosis [27].

In this study, we analyzed expression patterns of all the 44 mature miRNAs encoded by EBV in both serum and plasma samples by qRT-PCR. In order to prevent the influence of treatment factors, all the participants were ensured to be untreated when blood samples were drawn. In the primary selection phase, miRNAs with mean Ct-value $>$ 30 were excluded to guarantee adequate quantity of the identified circulating signature. As a result, only ebv-miR-BART19-3p was consistently up-regulated in the plasma of NPC patients compared to NCs (159 NPC VS. 145 NCs). The diagnostic performance of plasma ebv-miR-BART19-3p to distinguish NPC patients from NCs was assessed by ROC curve analysis. The AUC for the signature was 0.848 (95% CI: 0.807–0.889; sensitivity $=$ 71.7%, specificity $=$ 72.3%). Though the identified biomarker showed certain diagnostic value for NPC, no obvious association between miRNA expression level and NPC prognosis was discovered. Actually, due to the effectiveness of timely diagnosis and treatment, we could observe a favorable prognosis in the majority of NPC patients regardless of EBV miRNA expression levels in plasma.

miR-BART19-3p is a member of the miR-BART cluster that is highly expressed in epithelial tumors of NPC [50]. Up-regulation of miR-BART19-3p in NPC tissues and serum was once reported by Alissa et al. [27], but the sample size was very limited and its diagnostic potential was not validated specifically. However, they verified that miR-BART19-3p was one of the key regulators of the Wnt signaling pathway by targeting Wnt inhibitory genes such as WIF1, APC, and NLK [27, 51, 52, 53]. Constitutive activation of Wnt signaling can inhibit apoptosis and induce proliferation in various cancer cells including NPC [54, 55, 56]. Meanwhile, the anti-apoptotic effect of several miR-BART miRNAs in EBV-infected epithelial cells has also been proposed by a number of previous studies [57, 58, 59]. As supplement, we performed bioinformatics analysis to predict the potential target genes and pathways of miR-BART19-3p. A good deal of tumor-associated pathways such as Wnt signaling pathway, VEGF signaling pathway, and viral carcinogenesis were highly credible to be dysregulated by miR-BART19-3p, which could provide indication of EBV miRNA-related tumorigenesis.

To decipher the origin of circulating miR-BART19-3p, we further analyzed miRNA expression levels in 44 NPC tumor tissues in comparison with 32 unpaired normal tissues by qRT-PCR. Profound expressing difference in tissue samples could be observed. It was reasonable to consider that the up-regulated miR-BART19-3p in plasma could be derived from EBV-infected tumor cells or circulating tumor cells through active or passive transportation, the process of which might be deeply engaged in tumor progression. The consistent expressing characteristic of miR-BART19-3p in plasma and tissue samples further demonstrated the reliability of the identified biomarker for NPC detection.

Though differential expression of miR-BART19-3p was found in plasma samples, no significant difference was discovered in serum samples. In fact, the discrepancy of miRNA spectrum between serum and plasma has been previously observed [60]. One possible explanation for this discrepancy could be due to miRNA release or degradation during blood separation process [60, 61]. However, the serum and plasma samples involved in this study were not collected from the same individual at the same time. In addition, no suitable endogenous reference miRNAs were set up to normalize sample-to-sample variation. Therefore, paired samples with stable endogenous references were needed to elucidate the exact mechanisms.

Though most NPC cases are highly involved with EBV infection, in this study, we observed a considerable amount of NPC patients with cell-free EBV-DNA copy number less than 500 (regarded as EBV-negative). In fact, despite the promising sensitivity and specificity of circulating EBV copy number quantification for NPC screening, still a large proportion of NPC patients especially those in early disease stage or with precursor lesions are EBV load-negative [32, 41, 62, 63]. The possible explanation for this varying diagnostic accuracy could be due to the application of different PCR assays across laboratories, which lack in unified standards; DNA degradation in vivo and vitro for some technical or biological reasons could also limit its clinical use [64, 65]. In our present study, we observed relatively stable expression of the identified miRNA biomarker in both EBV-positive and EBV-negative patients. Unlike circulating EBV DNA molecules that are mostly released from apoptotic tumor cells, extracellular EBV-miRNAs can be actively capsuled into various carriers such as exosomes and lipoproteins, which protect them from degradation and ensure their stable expression in blood circulation [64, 66]. On the basis of our findings, we suspected that compared with EBV DNA, circulating EBV-encoded miRNAs might be more stable and detectable, if not with higher levels, to serve as diagnostic markers for NPC. At least, concomitant exploration of both circulating EBV DNA and EBV-derived miRNAs might help establish more promising indicators for NPC tumor phenotype. Meanwhile, no confounding effects of other clinical factors including tumor stage, age, and gender had been found in this study.

There are still some limitations in our study. For example, in-depth mechanism study of miR-BART-19-3p in NPC should be further conducted. Paired serum, plasma and tissue samples from the same participant will be better to compare miRNA expression patterns. In addition, microvesicles like exosomes can be further explored to decipher the potential origins of the up-regulated circulating miRNAs. Last but not least, no stable endogenous control miRNA had been established in this study.

Taken together, after comprehensive exploration both in serum and plasma samples, we identified a novel signature – miR-BART19-3p in plasma for NPC detection. Circulating miRNAs, with samples being easy to obtain and detecting means being cheap and fast, can have great advantages as tumor biomarkers. Hopefully, our work may benefit NPC detection in clinical application in the future.

Footnotes

Acknowledgments

This work was supported by the National Natural Science Foundation of China [Grant number: 81672400; 81770212].

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/CBM-190160.

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Circulating Epstein-Barr virus microRNA profile reveals novel biomarker for nasopharyngeal carcinoma diagnosis

Abstract

Keywords

1. Introduction

2. Materials and methods

2.1 Participants and characteristics of study cohort

2.2 Study design

Table 1 Demographic and Clinical characteristics of NPC patients and NCs

2.5 Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

2.6 Statistical analysis

3. Results

3.1 EBV miRNA expression profiling in plasma and serum samples

Table 2 Expression levels of miRNAs (mean Ct-value < 30) in serum and plasma samples. (presented as mean ± SD; Δ Ct, relative to cel-miR-39)

3.4 Subgroup analysis for various clinicopathologic features

3.5 Expression level of the identified EBV miRNA in tissue specimens

4. Discussion

Footnotes

Acknowledgments

Conflict of interest

Supplementary data

References

Table 1
Demographic and Clinical characteristics of NPC patients and NCs

Table 2
Expression levels of miRNAs (mean Ct-value $<$ 30) in serum and plasma samples. (presented as mean $\pm$ SD; $\Delta$ Ct, relative to cel-miR-39)