Serum lncRNA XIST has the Capacity to Serve as a Diagnostic Biomarker for Nasopharyngeal Carcinoma

Background LncRNA XIST (X-inactive specic transcript) is involved in various tumors. In this study, we aimed to investigate the diagnostic performance of serum lncRNA XIST in NPC. Serum samples were collected from 117 NPC patients and 80 gender and age matched healthy individuals. The relative expression of serum lncRNA XIST was detected by qRT-PCR. Chi-square test was applied to evaluate the relationship between lncRNA XIST expression and clinical characteristics. ROC analysis was performed to identify the feasibility of serum XIST as a diagnostic biomarker for NPC.


Background
Nasopharyngeal carcinoma (NPC) is one of the most common malignancies originating from the mucosal epithelium of the nasopharynx [1]. The incidence of NPC exhibits distinct regional diversity. Its morbidity is high in southeast Asia and Africa, particularly in southern China, while it is relatively rare in other regions of world [2][3][4]. The tumorigenesis of NPC is a complex process, with the involvement of genetic factors, environmental carcinogens, and Epstein-Barr virus (EBV) infection [5]. Like other types of malignancy, tumor stage at diagnosis is the key factor for outcomes of NPC patients. For patients diagnosed at early stage, the curve rate is high, however, the long-term survival remains dismal among those diagnosed with advanced stages [6]. Early diagnosis represents a great challenge in clinical setting, due its deep location and silent nature in early stage [7,8]. Until now, early detection of NPC mainly dependents on pathological biopsy and imaging. However, these methods are associated with several limitations, such as invasive, high cost, inconvenient, and time consuming, and they are not appropriate for routine screening and surveillance [6]. Consequently, it is urgently need to explore novel and effective diagnostic biomarkers to improve diagnosis and treatment of NPC.
Long non-coding RNAs (lncRNAs) are a group of RNAs with no or limited protein coding capacity which are longer than 200 nucleotides in length [9]. LncRNAs are implicated in various cellular processes, such as alternative splicing genes modi cation, histone modi cation, chromatin rearrangement and gene expression regulation [10]. Alteration of lncRNAs may contribute to human diseases, like cancer. Growing evidences have reported that lncRNAs played important roles in tumor initiation, progression and metastasis by regulating oncogenic and tumor-suppressing pathways in tumorigenesis [11]. LncRNA Xinactive speci c transcript (XIST), a product of XIST gene, is a newly identi ed lncRNA, and plays pivotal roles in regulating X inactivation in mammals [12]. It was reported that lncRNA XIST was associated with proliferation, differentiation, and genosome maintenance in tumor cells [13]. Dysregulation of lncRNA XIST was observed in diverse cancers, such as non-small cell lung cancer, breast cancer, glioblastoma carcinoma and ovarian cancer [13][14][15][16]. The function of lnc XIST in NPC was also reported in the previous studies. Song et al. suggested that XIST was up-regulated in NPC tissue specimens, and promoted the malignant progression of the cancer [17]. Based on the related researches, we deduced that serum XIST might be employed as a diagnostic biomarker for NPC.
In this study, the serum level of lncRNA XIST in NPC patients was detected, as well as its association with clinical characteristics. Additionally, we investigated the diagnostic performance of lncRNA XIST in NPC.

Patients and specimens collection
A total of 117 newly diagnosed NPC patients were recruited from Chinese PLA General Hospital. The diagnosis was based on pathological examination and tumors were classi ed according to the TNM cancer staging system (2009) set by the Union of International Cancer Control. 5 mL blood specimens were collected from NPC patients before receiving any treatments, such as radiotherapy or chemotherapy.
In addition, 80 gender and age matched healthy volunteers without any malignancies history were enrolled in the study, to serve as healthy controls. Blood specimens were also obtained from the healthy control group. Serum specimens were isolated from the blood samples via centrifugation, then the serum was stored at -80℃ until used. The basic data of the patients with NPC were recorded, including age, sex, family tumor history, clinical stage, TNM stage, and lymph node metastasis.
This study was approved by the Ethics Committee of Chinese PLA General Hospital. Each patient or family signed informed consent before blood collection.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from serum specimens using Trizol (Invitrogen) according to the manufacturer's instructions. The quality of RNA was evaluated by NanoDrop ND-1000 Spectrophotometer (Agilent, USA) and the RNA samples with the OD A260/A280 ratio close to a value of 2.0 were used for complementary DNA (cDNA) synthesis with the help of PrimeScript™ RT-PCR (Takara, Dalian, China). Quantitative realtime PCR (qRT-PCR) was performed to test the relative expression levels of lncRNA XIST. The reaction was performed using SYBR Premix Ex Taq™ (TaKaRa, Japan) in Applied Biosystems 7900 Fast Real-Time PCR system (Applied Biosystems, Foster City, California, USA). GAPDH acted as internal control, The primer sequences were as follows: XIST forward: 5'--CTCTCCATTGGGTTCAC-3', reverse: 5'-GCGGCAGGTCTTAAGAGATGAG-3'; GAPDH forward: 5'-TGCACCACCAACTGCTTAGC-3', reverse: 5'-GGCATGGACTGTGGTCATGAG-3'. The relative expression of XIST was normalized to GAPDH, and calculated by 2 −ΔΔCt method. Each experiment was repeated at least three times.
Statistical analysis SPSS 19.0 statistical software was applied for statistical analyses in the current study. The numerical data were presented as mean ± standard deviation (SD), and analyzed by Student's t-test. χ 2 tests was used to estimate the relationship between the expression of lncRNA XIST and clinicopathological characteristics. Receiver operating characteristic (ROC) analysis was performed to determine the diagnostic performance of serum lncRNA XIST in NPC. P value less than 0.05 was considered statistically signi cant.

Results
The expression level of lncRNA XIST 117 NPC patients including 71 men and 46 women were collected in our study. QRT-PCR was used to estimate the serum levels of lncRNA XIST. The result showed that serum lncRNA XIST was signi cantly higher in the patients with NPC than that in healthy volunteers (P < 0.001) (Fig. 1).

Relationship between lncRNA XIST and clinicopathological characteristics of NPC
In the current study, the patients were divided into high expression group (n = 68) and low expression group (n = 49), according to their mean serum level of XIST. Chi-square test was applied to estimate the potential association of XIST level with clinicopathological characteristics. Analysis results demonstrated that the expression of XIST was positively associated with advanced clinical stage (P = 0.013), TNM stage (P = 0.024) and lymph node metastasis (P = 0.003). However, no signi cant correlation was observed between the expression of lncRNA XIST and the age, gender, or family tumor history of the NPC patients (all P > 0.05) ( Table 1).  Fig. 2).

Discussion
Nasopharyngeal carcinoma (NPC) represents a malignant head and neck cancer which is characterized by highly invasive and metastatic [18]. Although the mechanisms underlying NPC remains unclear, it is general considered that NPC is affected by many factors, such as EB Virus infection, genetic alterations, dietary habit, as well as environmental factors [19,20]. Timely and appropriate treatments are pivotal for outcomes of NPC patients. Unfortunately, most NPC patients are diagnosed in advanced stage due to the silent nature of NPC in the early stage and lack of effective surveillance tools [8]. Diagnosis at metastasic stage may lead to high recurrence rate, therapeutic failures and poor prognosis [21]. Therefore, the sensitive and e ciency biomarker for early diagnosis are urgently needed to improve the treatments and prognosis of NPC patients.
LncRNA XIST (X-inactive speci c transcript) is a newly authenticated lncRNA deriving from XIST gene. Previous studies have proved that lncRNA XIST was involved in tumorigenesis based on its functional roles in cell proliferation, differentiation, and genome maintenance [22]. Alteration of XIST might lead to overall epigenetic instability, thus resulting in tumorigenesis [23]. For examples, the decreased expression of lncRNA XIST could contribute to the disappearance of the Barr body in breast cancer cells that might provide a new insight in the mechanisms of the disease [24]. Yildirim E et al. indicated that abnormal expression of lncRNA XIST was associated with X chromosome reactivation and overall changes of genome, thus promoting the progression of cancers [25]. Given its functional roles in tumorigenesis, lncRNA XIST was con rmed as a biomarker for several cancers. For example, the study carried out by Tantai and his colleagues demonstrated that serum lncRNA XIST might be employed as a diagnostic biomarker for non-small cell lung cancer patients [26]. Therefore, we deduced that lncRNA XIST might be a potential diagnostic biomarker for NPC.
In the present study, we studied the expression level of serum lncRNA XIST in NPC patients. Results suggested that the expression level of lncRNA XIST was up-regulated in NPC patients compared with that in healthy volunteers. Moreover, its elevated expression was signi cantly associated with clinical stage, TNM stage and lymph node metastasis. All the data revealed that lncRNA XIST as an oncogene was implicated in aggressive progression of NPC. It was reported that the oncogenic function of XIST in NPC might dependent on its regulatory role in expression of E2F3. XIST could up-regulate the expression of E2F3 via spongeing miR-34a-5p [17]. However, we did not investigate the speci c oncogenic mechanisms of lncRNA XIST in NPC in this study. Further researches were still needed.
Early diagnosis is key for outcomes of NPC patients. At present, the commonly used methods for NPC diagnosis include pathological biopsy and imaging. However, the clinical value of the methods are limited by invasive procedures, high cost, and time consuming [6]. In order to improve the diagnosis of NPC, various researches were devoted to explore novel and non-invasive biomarkers for screening of the cancer. A study of 529 subjects demonstrated that serum levels of cytokeratin 19 fragment 21.1 (CYFRA21-1) were signi cantly different between NPC patients and non-cancerous subjects that might be employed as a biomarker for the disease, moreover, its expression was closely correlated with EBV antibodies [27]. The predictive function of non-coding RNAs in NPC was also reported in the previous tumor investigation. For example, lncRNA actin lament associated protein 1 antisense RNA1 (AFAP1-AS1) was proved to play promoting roles in metastasis of NPC that might be a potential prognostic biomarker and therapeutic target for the cancer [28]. In this study, we estimated the diagnostic performance of lncRNA XIST in NPC. ROC curve showed that serum lncRNA XIST could distinguish NPC cases from healthy individuals with high sensitivity and speci city. Taken together, the altered molecules during tumorigenesis might provide novel biomarkers for early diagnosis and prognosis prediction of malignancy. Nevertheless, considering the relatively small sample size in the current study, well-designed researches with large sample size were still required to identify the results obtained in our study.

Conclusion
In conclusion, lncRNA XIST is up-regulated in NPC patients, and positively correlates with aggressive clinical characteristics. LncRNA XIST may be a potential diagnostic biomarker for NPC patients.

Disclosure
The authors report no con icts of interest in this work.

Author Information
Ethics approval and consent to participate This study was supported by the Ethics Committee of Chinese PLA General Hospital and also has been carried out in accordance with the World Medical Association Declaration of Helsinki.

Consent for publication
The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Data availability
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Competing interests
The authors declare that they have no competing interests. it. All authors read and approved the nal manuscript.