miR-639 is associated with advanced cancer stages and promotes proliferation and migration of nasopharyngeal carcinoma.

Early detection of nasopharyngeal carcinoma (NPC) is of vital importance for improving prognosis and survival rates. MicroRNA (miRNA) are a class of short and non-coding RNA molecules that are capable of inhibiting the translation of mRNA of target genes. Previous studies have revealed that miRNA are involved in tumorigenesis and cancer development. The RNase-resistance of circulating miRNA have made them valuable non-invasive biomarkers, and has therefore drawn particular attention to their therapeutic potential. The aim of the present study was to investigate the expression of the previously uncharacterized miR-639 in NPC. In a study population of 139 patients, higher expression of miR-639 was associated with metastasis, more advanced cancer stages, and lower disease-free survival rates. In vitro experiments involving transfection of human NPC C666-1 and NPC/HK1 cell lines with miR-639 mimics and antagomir indicated that overexpressing miR-639 promoted cell proliferation and migration, suppression of miR-639 inhibited proliferation and migration. The present study provides evidence that miR-639 is differentially expressed in NPC tissues of varying cancer stages, and suggests that quantifying circulating miR-639 may be of importance for non-invasive diagnosis and prognostic evaluation, and may have potential therapeutic utility.

Introduction

Nasopharyngeal carcinoma (NPC) is a type of malignancy that occurs frequently in Southeast Asia, particularly in the Chinese population (1,2). It is often diagnosed at a late stage and the prognosis remains poor (3,4). Although substantial improvements have been made in treating NPC, particularly with significant improvements in survival rate using intensity-modulated radiology (5), management of this disease remains an issue (6). For the previous two decades, studies have been aimed at identifying biomarkers for NPC diagnosis and prognosis, and these biomarkers have presented valuable information. However, these markers require invasion to tumor tissues, which has made their use inconvenient, and has limited their application. Therefore, biomarkers applicable for non-invasive diagnosis are required for prognosis and treatment evaluation in this disease. Previously, RNase-resistant circulating microRNA (miRNA) have gained attention, and were frequently identified as a promising component for methods of early detection and prediction of outcomes of different types of cancer (7).

miRNA are 19–25 nucleotide-long endogenous noncoding RNA that serve as critical components in regulating gene expression (8). They downregulate the expression of target genes via sequence-specific interactions with the 3′untranslated region (UTR) of cognate mRNA targets, which will subsequently be degraded or inhibit the translation efficiency of mRNA (9). As embryonic development and human diseases are closely associated with abnormalities in miRNA expression profiles, differentially expressed miRNA have gained attention. Various previous studies have suggested that miRNA serve a pivotal role in cell proliferation, migration, differentiation, apoptosis, cell cycle and angiogenesis, all of which are key biological processes in tumorigenesis and cancer development (10–13). According to previous estimation, ~50% of annotated human miRNA are detected in tumor tissues, including NPC (14,15). Previous evidence has also indicated that subsets of miRNA hold promise as diagnostic and prognostic biomarkers in various types of malignancies (16).

The differential expression of miR-639 has only been detected in breast cancer and tongue squamous cell carcinoma, but has yet to be determined in NPC (14,17). Particularly, previous studies have suggested that a reduced expression of miR-639 is associated with transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition in tongue squamous cell carcinoma by targeting Forkhead Box C1, indicating potential value in therapies for metastasis targeting these processes (17). These data were additionally supported by a study demonstrating an elevated expression of miR-639 in patients with metastasis compared with those without metastasis (14). In light of this, the present study examined the expression of miR-639 in NPC tissue samples. It was identified that miR-639 expression was increased in metastatic NPC tissues, and that miR-639 mimics increased the invasion of NPC cell lines. Furthermore, associations between miR-639 and advanced tumor stages, and poor survival were demonstrated. These results reiterate the important role of miR-639 in NPC, and suggest that exogenous overexpression of miR-639 promotes invasion and migration in NPC.

Materials and methods

Sample source

All samples were obtained via biopsy from The First Affiliated Hospital of Nanjing University (Nanjing, China), which were ascertained as NPC at the time of diagnosis. Briefly, immunohistochemical staining was performed on deparaffinized tissue sections (4–5 µm) of formalin-fixed material following microwave-enhanced epitope retrieval. Detection was performed with streptavidin-horseradish peroxidase (cat. no., 405210; BioLegend, Inc., San Diego, CA, USA), with 3-amino-9-ethyl carbazole as the chromogen and hematoxylin for counterstain, modified according to Gill III (80 g/l aluminum sulfate, 0.6 g/l sodium iodate, 6 g/l hematoxylin, 0.25% ethylene glycol and 0.02% glacial acetic acid), for 3 min at room temperature. The recruitment was from March 2013 to July 2015, during which follow-up was conducted on these patients (Table I). The tissues were immediately frozen in liquid nitrogen and stored at −80°C, and the medical records of patients were carefully reviewed. Informed consent was obtained for all patients in the present study, which was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing University (Jiangsu, China).

Table I.

Associations between miR-639 expression and clinical parameters (n=139).

Parameters	No. of patients, n (%)	miR-639 ΔCq (mean ± standard deviation)	P-value
Age, years
≤40	36 (25.9)	1.83±0.16	0.408
41–50	53 (38.20)	1.76±0.23
51–60	27 (19.4)	1.86±0.18
>60	23 (16.5)	1.79±0.21
Sex
Men	94 (67.6)	1.81±0.22	0.523
Women	45 (32.40)	1.78±0.20
Histologic features			0.418
WHO Type 2	47 (33.8)	1.78±0.24
WHO Type 3	92 (66.2)	1.81±0.20
AJCC 6th Stage			1.009×10-4
I	7 (5.0)	1.42±0.05
II	33 (23.7)	1.79±0.24
III	45 (32.4)	1.84±0.20
IV	54 (38.9)	1.83±0.18
M stage
0	118 (84.9)	1.77±0.21	0.016×10-2
1	21 (15.1)	1.96±0.17

WHO, World Health Organization; AJCC, American Joint Committee on Cancer.

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Samples of NPC tissue and adjacent normal nasopharynx tissue were obtained from patients undergoing surgery and were snap frozen in liquid nitrogen and stored at −80°C following surgical resection. Extraction of RNA was performed using Qiagen RNeasy Mini kits (cat. no. 74104; Qiagen China Co., Ltd., Shanghai, China) according to the manufacturer's protocol. RNA was purified using a Qiagen RNase free DNase kit (cat. no. 79254; Qiagen China Co., Ltd.). The purity and concentration of total RNA was determined using a spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the optical density (OD)260/OD280 ratio between 1.8 and 2.0 was considered as high purity. The miRcute miRNA first strand cDNA Synthesis kit (cat. no. KR201; Tiangen Biotech Co., Ltd., Beijing, China) was used to reverse transcribe cDNA of miR-639, and the miRNA quantification was performed by miRcute miRNA qPCR detection kit (cat. no. FP401; Tiangen Biotech Co., Ltd.), according to the manufacturer's protocols. U6 small nuclear RNA (U6-snRNA) purchased from Takara Biotechnology Co., Ltd. (Dalian, China) was used as an internal control. For miRNA quantification, the following PCR conditions were used: 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec and 60°C for 1 min, and a dissociation stage at 60°C for 10 min. The threshold cycle (Ct) is defined as the cycle number at which the change of fluorescence intensity crosses the threshold. The relative expression levels were calculated as 2−[(Ct of miR-639)-(Ct of U6)] following normalization to U6 small nuclear RNA. The primer sequences used in the qPCR were: miR-639, forward, 5′-ATCGCTGCGGTTGCGAGCGCTGT-3′ and U6, forward, 5′-GGGCAGGAAGAGGGCCTAT-3′.

Cell culture and transfection

The NPC C666-1 and NPC/HK1 cell lines were purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences Cell Bank (Shanghai, China). RPMI-1640 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) medium supplemented with 5% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to culture the C666-1 and NPC/HK1 cells, which were maintained at 37°C in an atmosphere with 5% CO2. Three days following transfection, cells were harvested for subsequent experiment.

Subsequent to reaching 70–80% confluence, the C666-1 and NPC/HK1 cells were transfected with 50 nM miR-639 mimics (5′-AUCGCUGCGGUUGCGAGCGCUGU-3′), antagomir (5′-ACAGCGCUCGCAACCGCAGCGAU-3′) or scramble sequences (5′-UCACAACCUCCUAGAAAGAGUAGA-3′) provided by Thermo Fisher Scientific, Inc., for 24 h at room temperature with Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

MTT cell viability assay

Cells were seeded in 96-well plates at a cell density of 1×104 cells/well. MTT solution (20 µl of 5 mg/ml for each well) was added to cultures, which were then incubated at 37°C for 4 h. Subsequent to removing the culture medium, the crystals were dissolved in 3% dimethyl sulfoxide, the absorbance at 570 nm of which were then measured using a spectrophotometer (Bio-Rad Laboratories, Inc.).

Scratch wound healing assay

C666-1 and NPC/HK1 cells were grown in RPMI-1640 supplemented with 10% FBS to 70–80% confluence. Following transfection with miR-639 mimics, antagomir or scramble control for 24 h, a 200-µl pipette tip was used to make a straight scratch on the culture, followed by washing with Hanks medium (Beyotime Institute of Biotechnology, Haimen, China) until no floating cells were present. The scratched cultures were then maintained in serum-free RPMI-1640 media (Thermo Fisher Scientific, Inc.). Images were captured immediately and at 24 h using the Zeiss AxioCam MR digital camera on a Zeiss Axioskop2 light microscope (magnification, ×200; Carl Zeiss AG, Oberkochen, Germany). The remaining wound area was measured using ImageJ 1.50 g software (National Institutes of Health, Bethesda, MD, USA) and normalized to the original wounds.

Statistical analysis

All statistical analysis was performed using SPSS (version 22; IBM Corp., Armonk, NY, USA). Each assay was performed in triplicate, and all values were expressed as the mean ± standard deviation. The statistical significance of differences between groups was evaluated by one-way analysis of variance followed by Dunnett's test. Kaplan-Meier analysis was used to analyze survival data of increased miR-639 expression decreased miR-639 expression. P<0.05 was considered to indicate a statistically significant difference.

Results

Association of miR-639 expression with metastasis of NPC

Tissue specimens from 139 patients with NPC were gathered in total, the details of which are summarized in Table I. RT-qPCR was performed to measure the expression level of miR-639 in a group of tissue specimens from patients with NPC. In the 21 metastatic NPC tissues, the expression level of miR-639 was 1.96±0.17, while in the 118 non-metastatic NPC tissues the expression level of miR-639 was 1.77±0.21 (P=0.00016; Fig. 1A). These results demonstrated that the miR-639 expression level was increased in primary metastatic NPC tissues compared with the level in non-metastatic NPC tissues. The association between miR-639 expression levels and clinical parameters was then evaluated. miR-639 expression level was identified to be significantly associated with more advanced cancer stages (P=0.0000109; Table I).

Figure 1.

Association between miR-639 expression level and metastasis and survival rates. (A) Engelmann-Hecker plot of relative expression level of miR-639 in M1 and M0 tissues. In 21 M1 tissues, the average expression of miR-639 is significantly increased compared with that of M0 (P=0.00016). (B) Disease-free survival rates of high miR-639 expression group and low miR-639 expression group. The x-axis represents survival time and y-axis represents disease-free survival rate, the difference in survival rate between the two groups was significant (P=0.0236). M0, non-metastatic tissues; M1, metastatic tissues; miR, microRNA.

Kaplan-Meier disease-free survival analysis was then conducted based on the expression level of miR-639. The samples were divided into two groups according to the mean expression level: High miR-639 expression (≥1.8) and low miR-639 expression (<1.8). The low expression group demonstrated a significantly (P=0.0236) longer survival rate compared with the high expression group (Fig. 1B). These results indicate that miR-639 expression is associated with cancer progression of NPC.

miR-639 overexpression promotes proliferation of NPC cell line C666-1 and NPC/HK1 cells

Provided that the differential expression of miR-639 between metastatic and non-metastatic NPC tissues has been observed, indicating the potential role of miR-639 in cancer progression, the biological function of miR-639 in NPC proliferation was investigated by introducing miR-639 mimics to NPC C666-1 and NPC/HK1 cell lines. MTT assay was performed to measure rate of cell growth, and the expression level of miR-639 was determined using RT-qPCR (Fig. 2A). In C666-1 and NPC/HK1 cells, transfection of miR-639 mimics significantly increased the cell viability whereas transfection of miR-639 antagomirs reduced cell viability. C666-1 and NPC/HK1 cells overexpressing miR-639 consistently demonstrated increased cell growth compared with those transfected with scramble sequences. In addition, those cell lines transfected with an antagomir of miR-639 was identified to inhibit cell proliferation (Fig. 2B and C). These results suggest that miR-639 may promote proliferation of NPC cells in vitro.

Figure 2.

miR-639 expression promotes proliferation of C666-1 and NPC/HK1 cells. (A) Relative expression level of miR-639 in C666-1 and NPC/HK1 cells transfected with miR-639 mimics, antagomirs and scramble control. (B) MTT cell viability assay of C666-1 cells transfected with miR-639 mimics, antagomirs and scramble control. (C) MTT cell viability assay of NPC/HK1 cells transfected with miR-639 mimics, antagomirs and scramble control. *P<0.05 and **P<0.01 vs. scramble. Error bars indicate the standard deviation. OD, optical density; miR, microRNA.

Overexpression of miR-639 promotes metastasis of NPC

As aforementioned, the present study demonstrated that miR-639 is associated with metastasis in patients with NPC, and that it promotes the proliferation of NPC cells. However, additional direct evidence confirming its causal role in the aggressive traits of NPC are required. Therefore, the same cell lines transfected with miR-639 mimics and antagomirs were used to investigate whether miR-639 also enhances the invasive capacity and metastasis of NPC. Scratch wound healing and scratch wound healing assays were conducted in C666-1 and NPC/HK1 cells to evaluate the invasion capacity and metastasis, respectively. In the scratch wound healing assay presented in Fig. 3A and B, C666-1 and NPC/HK1 cells overexpressing miR-639 exhibited significantly higher invasion capacities compared with their respective controls (P=0.034 and P=0.0256, respectively). In the scratch wound healing assay, similar differences were also observed between those transfected with miR-639 and the controls. Conversely, downregulation of miR-639 by transfection with miR-639 antagomir led to an inhibition of invasion and metastasis abilities in the two cell lines.

Figure 3.

(A) Quantification and (B) microscopic images (magnification, ×200) of scratch wound healing assays of C666-1 and NPC/HK1 cells transfected with miR-639 mimics, antagomirs and scramble control. **P<0.01 vs. scramble. miR, microRNA.

Discussion

NPC is an epithelial malignancy of the uppermost portion of the pharynx, which exhibits distinct geographical, etiological and biological features (18). NPC occurs behind the nasal cavity, the affected area of which extends from the upper surface of the soft palate to the base of the skull. The high prevalence of NPC is specific to distinct endemic regions such as Southeast Asia, where the incidence is up to 20–50/100,000 person-years (12–14). Previous studies have revealed a number of human nucleic acids, protein and metabolite biomarkers that demonstrate potential for diagnostic and prognostic evaluation. These biomarkers include the hypermethylated promoters of known tumor suppressor genes, including death-associated protein kinase (19), cyclin-dependent kinase inhibitor 2A (20) and cadherin 1 (21), and critical cytokines including TGF-β, macrophage inflammatory protein-3α (22), cluster of differentiation 40-ligand (23), and interferon-γ (24). Previously, circulating miRNA have gained extensive attention and exhibited great potential as they are resistant to RNase degradation (25,26).

According to miRNA-mRNA complementarity, miRNA may either cause mRNA degradation or suppress mRNA translation. Argonaute 2 is a ‘slicer’ protein that mediates the degradation of target mRNA when the complementarity is perfect or near-perfect. While in imperfect complementarity, miRNA-mRNA together with all four Argonaute proteins form a complex called ‘miRISC’, which is capable of inhibiting protein translation (27). In essence, the net effect of miRISC binding to a target mRNA is a decrease in its protein levels. However, previous studies have demonstrated a small number of exceptions, wherein protein expression is actually increased: For example, miR-10A may increase the expression of several ribosomal proteins through binding to the 5′UTR of their mRNA transcripts (28). Despite the complicated regulation of miRNA, the effect of miRNA on tumorigenesis and cancer development suggests its important role in molecular mechanisms underlying tumorigenesis and cancer development. For instance, MYC may induce the expression of miR-17/92, and miR-17/92 increases the anti-apoptosis activity of E2F1, enhancing the proliferation of cancer cells (29). In contrast, certain miRNA that are inhibited by MYC, including let-7, miR-34a and miR-15a/16-1, have frequently been identified as tumor suppressors (30–32).

To the best of our knowledge, the present study demonstrated for the first time that miR-639 is differentially expressed in metastatic and non-metastatic NPC tissues. The average miR-639 expression level in metastatic NPC tissues was higher than in non-metastatic NPC tissues and, consistent with this, the higher expression of miR-639 was associated with more advanced cancer stages and lower survival, indicating the potential prognostic and diagnostic utility of miR-639. To identify the role of miR-639 in tumorigenesis and cancer development, miR-639 mimics and miR-639 antagomirs were transfected into C666-1 and NPC/HK1 cell lines. Proliferation and migration were identified to be promoted by miR-639 mimics, but diminished by miR-639 antagomirs. The observations in the two cell lines are consistent, suggesting that miR-639 may be a common biomarker for different subtypes of NPC. Consistent with this, some researches have reported that miR-639 could promote proliferation in other types of cancer cells (33,34). However, a number of studies have reported the tumor suppressive function of miR-639 (35,36). This contradiction can be explained by the heterogeneity of cancer, as miRNAs have multiple targets, in which some serve as oncogenes, while others may serve as tumor suppressors. Therefore, the effect of miRNAs on cancers is determined by the overall effect of these aforementioned targets.

The abnormal expression of miR-639 has been demonstrated in urinary bladder cancer, the vitreous humor of patients with ocular diseases, breast cancer and tongue cancer (14,17,37,38). As miRNA function through interaction with target mRNA, the identification of target genes of miR-639 is important for exploring the mechanism underlying its role in NPC. Previously, Sandip et al (39) conducted expression profiling of miRNA of the human breast cancer MCF-7 cell line deprived of breast carcinoma amplified sequence, and identified that miR-let-7b, miR-139-3p, miR-4720-5p, miR-486-3p, miR-589 and miR-639 were aberrantly expressed. TargetScan 6.2 and miRanda have predicted that CAT-201, protein tyrosine phosphatase, receptor type J and cytochrome P450 family 11 subfamily B member 2 are potential targets of miR-639, which had been documented as being associated with the risk of breast cancer in multiple studies (40–43). In addition, Wu et al (44) revealed that miR-639 may reduce the expression of p21Cip1/Waf1, a cyclin-dependent kinase inhibitor implicated in the response of cells to genotoxic stress and a major transcriptional target of tumor protein 53 protein. These putative mechanisms warrant experimental validation, and may allude to directions for future studies investigating the role of miR-639 in NPC.