|Year : 2015 | Volume
| Issue : 4 | Page : 111-114
Expression Characteristics of miR-10b in Nasopharyngeal Carcinoma
Gang Li, Yunteng Zhao, Jianqi Wang, Haoran Huang, Mengwen Zhang
Department of Otolaryngology, Head and Neck Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
|Date of Submission||11-Jul-2015|
|Date of Acceptance||12-Aug-2015|
|Date of Web Publication||27-Aug-2015|
Dr. Gang Li
Department of Otolaryngology, Head and Neck Surgery, Nanfang Hospital, 1838 Guangzhou Dadao Bei, Southern Medical University, Guangzhou 510515, Guangdong
Source of Support: This study was supported in part by grants from the National
Natural Science Foundation of China to Gang Li (No. 81472534), Conflict of Interest: None
Aim: Nasopharyngeal carcinoma (NPC) is a highly malignant neoplasm characterized with aggressive local invasion and strong tendency of distant metastasis. MicroRNAs (miRNAs) are small noncoding RNAs that play a critical role in the pathogenesis of various malignant tumors. Our previous study aims to explore miRNAs' expression characteristics in NPC tissue, which is barely unknown.
Methods: A total of 45 NPC samples (26 metastatic and 19 nonmetastatic) and 16 chronic nasopharyngitis samples were analyzed. miR-10b level was determined by in situ hybridization using digoxigenin-labeled locked nucleic acid-based probe, and latent membrane protein-1 (LMP-1) expression were detected by immunohistochemistry.
Results: We show that miR-10b is differentially expressed in NPC tissues. Furthermore, we correlate the expression level of miR-10b with Epstein-Barr virus encoding LMP-1. Overexpression of miR-10b is also found to relate with the clinical phases of NPC.
Conclusion: These findings strongly suggest that miR-10b plays an important role in the NPC development.
Keywords: Latent membrane protein-1, metastasis, microRNAs, nasopharyngeal carcinoma
|How to cite this article:|
Li G, Zhao Y, Wang J, Huang H, Zhang M. Expression Characteristics of miR-10b in Nasopharyngeal Carcinoma. Cancer Transl Med 2015;1:111-4
|How to cite this URL:|
Li G, Zhao Y, Wang J, Huang H, Zhang M. Expression Characteristics of miR-10b in Nasopharyngeal Carcinoma. Cancer Transl Med [serial online] 2015 [cited 2019 Dec 9];1:111-4. Available from: http://www.cancertm.com/text.asp?2015/1/4/111/163801
| Introduction|| |
Geographically, nasopharyngeal carcinoma (NPC) is most prevalent in Southeast Asia, with particularly a high incidence in Southern China. NPC is distinctive among the head and neck carcinomas for its marked tendency to metastasize and invade.  It has been shown that by the time of diagnosis, 60-85% of NPC patients already have a clinically detectable aggressive metastasis in the regional lymph nodes and in distant organs such as lungs and even in bone. So far, there are still no effective treatments available for NPC, especially at the stage of metastasis. Therefore, prognosis of NPC is considerably poor, with a 5-year survival rate in < 50% of the cases.  Meanwhile, the mechanisms regulating NPC metastasis remain poorly understood.
Epstein-Barr virus (EBV) encoding latent membrane protein-1 (LMP-1) is a primary oncoprotein in NPC. It has been attributed to the highly metastatic nature of NPC. LMP-1 functions as a constitutively active tumor necrosis factor receptor while contributing to multiple aspects of NPC, mainly through activating a number of signaling pathways, including nuclear factor kappa, activator protein 1, ets-1, mitogen-activated protein kinases, and JAK/STAT, and controlling the expression of metastasis related gene such as E-cadherin, matrix metalloproteinase, c-Met, vascular endothelial growth factor, epidermal growth factor receptor, and cyclooxygenase-2. Under in vitro conditions, knocking down the LMP-1 has shown to diminish the metastatic abilities of NPC cells. 
In our previous study, we found that microRNA (miRNA)-10b (miR-10b) initiated the NPC invasion and metastasis. We also found that Twist, a metastasis promoting transcription factor, could induce miR-10b expression and demonstrated that miR-10b was an essential element in the Twist-induced metastasis program.  However, we still do not know if miR-10b gene could be considered as a therapeutic marker for NPC metastasis in patient tissue sample.
Therefore, we analyzed the miR-10b expression in NPC patients' tissue samples. For the 1 st time, we confirmed the increased expression of miR-10b in human NPC tissue and it's correlation with LMP-1.
| Methods|| |
Patients and samples
This study was conducted on a total of 45 NPC samples (26 metastatic and 19 nonmetastatic) and 16 chronic nasopharyngitis samples. All the patients were diagnosed through endoscopic biopsy from June 2012 to December 2013 in Nanfang Hospital, China. All the NPC patients underwent complete examination which included magnetic resonance imaging of the nasopharynx, skull base and neck, chest-X-rays, abdominal ultrasonography, and Single-photon emission computed tomography bone scan. For patients with clinical evidence of distant metastases, positron emission tomography-computed tomography was employed for evaluation. All the NPC patients were carefully classified according to 2008 edition of the AJCC-American Joint Committee on Cancer staging criteria, and received an image-guided intensity modulated radiotherapy. For patients staging at and over T2, or with lymphatic metastasis, cisplatin-based concurrent chemotherapy following induction chemotherapy were given, according to National Comprehensive Cancer Network Guidelines (Version V.2.2 2008). NPC patients were requested for follow-up schedules once every 3 months, after complete initial treatment, in the first 2 years and every 6 months thereafter. Informed consent was obtained from all the patients, and research protocol was approved by the Ethics Committee of Nanfang Hospital.
All the samples were divided into two parts, of which one part was frozen and cryosectioned for in situ hybridization (ISH) to localize miR-10b, and the other was paraffin-sectioned for the immunohistochemical analysis of LMP-1.
In situ hybridization
miR-10b level was determined by ISH using digoxigenin (DIG)-labeled, locked nucleic acid (LNA)-based probe, specific for miR-10b (Exiqon, Vedbaek, Denmark), according to the manufacturer's protocol. U6 small nuclear was used as a positive control. Briefly, frozen sections were treated with proteinase K (5 min in 2 mg/mL proteinase K), washed in phosphate buffered saline (PBS) and subsequently blocked the endogenous peroxidase activity with 3% H 2 O 2 . Hybridization was performed at 52°C overnight, after the addition of 50 nM of DIG-labeled LNA probes, followed by a stringent wash in saline sodium citrate buffers. After blocking (2% sheep serum and 2 mg/mL BSA in PBS with Tween-20) at room temperature, the probe-target complex was visualized with an anti-DIG-POD antibody and 3,3'- diaminobenzidine (DAB) complex.
LMP-1 expression in the NPC tissues was detected by immunohistochemistry. All the tissues were fixed in neutral buffered formalin and then embedded in paraffin before sectioning, at a thickness of 4 mm. After deparaffinizing, the sections were rehydrated through gradient alcohol and PBS. Slides were then placed in Tris/ethylenediaminetetraacetic acid (pH 9.0) buffer and heated for 6 min in a microwave oven, followed by boiling in a pressure cooker, immediately. As soon as the cooker reached full pressure, time of 4 min, it was turned off and allowed to cool under running water, till the buffer returned to room temperature. The slides were then treated with 3% H 2 O 2 in methanol for 15 min, blocking endogenous peroxidase activity. After blocking nonspecific antigens with 5% goat anti-human serum, for 1 h at room temperature, 20 mL of mouse anti-LMP-1 monoclonal antibody (CS1-4 Dako), at 1:100 dilution, was added to each slide and incubated at 4°C overnight. Later, the slides were incubated with a mixture of goat biotinylated anti-mouse IgG and IgM and anti-rabbit IgG antibodies for 1 h, and then with horseradish peroxidase-conjugated streptavidin for 30 min. The sections were thoroughly washed with PBS among the steps. Visualization was achieved by treating the slides with DAB solution for 3 min. Finally, slides were routinely counterstained with Gill's hematoxylin and dehydrated before mounting and stored at 4°C. The staining density was analyzed using (Media Cybernetics, Inc., Silver Spring, MD, USA). The relative expression of LMP-1 is defined as integrated option density/SUM AREA (IOD/S) IOD/S.
All analyses were carried out using SPSS17.0 (SPSS Inc., Chicago, IL, USA) software package. Expression of miR-10b, in relation to clinical data, was analyzed with the Mann-Whitney U test. Comparison of mean was analyzed by one-way ANOVA and Student-Newman-Keuls method for multiple comparisons. Dunnett's T3 method was employed for the heterogeneity of variance. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. The χ2 test for proportion was used to analyze the in vivo metastasis characteristics of different groups. A P value less than < 0.05 was considered as statistically significant.
| Results|| |
miR-10b expression in nasopharyngeal carcinoma tissues and control group
miR-10b expression pattern was explored by ISH in 45 NPC samples (26 metastatic and 19 nonmetastatic) and 16 chronic nasopharyngitis samples. There was no notable difference seen in the miR-10b expression between NPC (36/45) and chronic nasopharyngitis (9/16) samples (χ2 = 3.441, P = 0.097) [Table 1].
miR-10b was over-expressed in metastatic nasopharyngeal carcinoma samples
ISH results showed lower miR-10b expression in nonmetastatic NPC samples. The results also showed a positive expression of mir-10b in both NPC and chronic nasopharyngitis tissue samples, with its localization in both nucleus and cytoplasm of the cells [Figure 1]. The expression was also found to be significantly increased in the metastatic patients' tissues compared to nonmetastatic NPC cells (Z = −3.243, P = 0.001) [Table 2].
|Figure 1: Identification of miR-10b expression in the nasopharyngeal carcinoma tissues and chronic nasopharyngitis tissues by in situ hybridization. (a) Negative expression in chronic nasopharyngitis; (b) positive expression in chronic nasopharyngitis, located in the nucleus and cytoplasm; (c) negative expression in nasopharyngeal carcinoma; (d) weak expression in nasopharyngeal carcinoma (+); (e) positive expression in nasopharyngeal carcinoma (++), located in the nucleus and cytoplasm; (f) extremely strong expression in nasopharyngeal carcinoma (++++). 3,3'-diaminobenzidine staining and hematoxylin counterstaining, ×400|
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miR-10b was correlated with latent membrane protein-1 expression
Furthermore, the expression of miR-10b was correlated with LMP-1 expression (Z = −2.013, P = 0.044) [Figure 2] and [Table 3]. An increased levels of miR-10b were seen in LMP-1(+) metastatic NPC tissues than nonmetastatic samples (Z = −2.504, P = 0.012) while miR-10b was maintained at its low expression levels in LMP-1(−) cases. Also, no significant difference was seen between the metastatic and nonmetastatic subgroup (Z = −1.857, P = 0.063) [Table 4].
|Figure 2. Expression of latent membrane protein-1 in nasopharyngeal carcinoma (streptavidin-biotin complex method of immunohistochemistry, 3,3'-diaminobenzidine staining, ×200). (a) Positive expression, located in the membrane; (b) negative expression|
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|Table 3: Relationship between LMP-1 and miR-10b expression in NPC tissue |
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|Table 4: Relationship between NPC metastasis and miR-10b in LMP-1 (+) or (−) NPC tissue |
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| Discussion|| |
MiRNAs are 19-25 nt regulatory RNAs that participate in the regulation of various biological functions in numerous eukaryotic lineages, including plants, insects, vertebrate, and mammals. miRNAs are integral to gene regulation, apoptosis, hematopoietic development, and maintenance of cell differentiation. There are a growing number of examples of particular miRNAs which are increasingly expressed in cancer (such as miR-21, the miR-17-92b cluster, miR-155, and miR-372/373) whereas the expression of a few others get decreased in tumor cells, as compared to normal cells. miRNAs that have been experimentally shown to directly induce tumor have been termed as "oncomirs," with the first example being the mir-17-92b cluster.  Metastasis is a crucial problem in cancer, yet the underlying mechanisms remain poorly understood. The involvement of miRNAs in the development of metastases has been gained attention and is under intense scrutiny, in the recent years. miR-10b was proved to be involved in different cancer metastasis. ,,, Ma et al.  reported that anti-miR-10b treatment alone decreased invasiveness of breast cancer cell by 90%, and the same was proposed to be a new preventive measure against the spread of deadly tumors. ,
In our previous study, we showed that LMP-1 could activate miR-10b, possibly via Twist, which in turn stimulated cell migration and invasion, under in vitro conditions. We later showed that in cell culture, blocking miR-10b could directly inhibit the invasive behavior of miR-10b over-expressed NPC, but such effect was not found in the control cells without miR-10b over-expression.  We established a NPC metastasis model in mice by transplanting miR-10b over-expressed EBV-positive NPC cells, C666/shLMP-1/miR-10b, in the livers of nude mice and observed lung metastasis. This metastasis was not found when miR-10b over-expressed EBV-negative nonmetastatic NPC cells were transplanted into mice. Other works have also confirmed that the induction of Twist by EBV-encoded with human viral oncoprotein LMP-1 directly contributes to the metastatic nature of NPC cell. 
In this study, we further investigated the expression characteristics of miR-10b in NPC tissues for the 1 st time. Our data indicate that miR-10b is up-regulated in the metastatic NPC, and its expression level is positively correlated with LMP-1. LMP-1 contributes to the multiple steps of the signal cascade, in NPC, through numerous downstream molecules,  of which miR-10b pathway is likely to be one of such metastasis promoters. However, no difference in miR-10b expression between NPC and chronic nasopharyngitis was observed in our study, which implied that miR-10b promoted NPC aggressiveness, but did not play an essential role in the initiation of the disease.
These results also indicated that multiple mechanisms may display in the development of NPC that increase our difficulties to identify specific target for molecular targeted therapy.
Financial support and sponsorship
This study was supported in part by grants from the National Natural Science Foundation of China to Gang Li (No. 81472534).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]