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1 DOI /s ORIGINAL ARTICLE Upregulation of mir-21 and downregulation of mir-494 may serve as emerging molecular biomarkers for prediagnostic samples of subjects who developed nasopharyngeal carcinoma associates with lymph node metastasis and poor prognosis Masoomeh Dadpay 1 & Mojtaba Zarea 2 & Rahman Ghaffarzadegan Rabati 3 & Bijan Rezakhaniha 4 & Babak Barari 5 & Vahid Behnod 6 & Katayoun Ziari 7 Received: 27 June 2015 /Accepted: 5 August 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015 Abstract The aim of this study was to investigate the expression level of mir-21 and mir-494 in nasopharyngeal carcinoma (NPC) patients. Real-time PCR was applied to quantify the expression level of mir-21 and mir-494. Moreover, the correlation of these markers with pathological characteristics was also evaluated in NPC cases. Our result revealed that mir-21 expression was higher in tumor tissue when compared with normal tissues. Moreover, downregulation of mir-494 was detected in NPC patients when coppered with normal nasopharyngeal tissues. The results recommended that high expression of mir-21 and low expression of mir-494 were strongly associated with high tumor-node-metastasis (TNM) stage (P=0.023; P=0.016) and lymph node metastasis (P= 0.031; P=0.041). Nevertheless, there was no significant relationship of mir-21 and mir-494 expression with other clinical factors including age (P=0.621; P=0.561), sex (P=0.547; * Katayoun Ziari drk_ziari@yahoo.com Department of Pathology, Imam Reza Hospital, AJA University of Center for Chemical Biology, Indian Institute of Chemical Technology (IICT), Tarnaka, Hyderabad, India Shahid Abbas Abdollahi, Molecular Biology Research Center, Shahid Mahallati Hospital, Tabriz, Iran Department of Urology, Imam Reza Hospital, AJA University of Dr Genetic Medical, Tehran University of Medical Sciences, Tehran, Iran Baqiyatallah University of Department of Pathology, Be sat Hospital, AJA University of P=0.634), degree of differentiation (P=0.426;P=0.301), histology (P=0.321; P=0.432), and distal metastasis (P=0.582; P=0.472). Survival analysis and log-rank test revealed that patients with high expression of mir-21 had shorter overall survival in comparison with patients who had low mir-21 expression (log-rank test P=0.002). In addition, low rate of mir-494 expression was associated with shorter time survival than patients when compared with high expression group of patients (log-rank test P=0.024). Multivariate Cox model indicated that high expression of mir-21, low expression of mir-494, and TNM stage were independently associated with poor survival of NPC patients. Our data showed that upregulation of mir-21 was linked to poor prognosis of NPC patients. On the other hand, downregulation of mir-494 may play a crucial role as independent prognostic factor in NPC. Keywords mirnas. Survival. Patient. Clinicopathological. Nasopharyngeal carcinoma Introduction MicroRNAs (mirnas) are small noncoding RNA molecule, which play a key role in the regulation of gene expression. mirnas are involved in many biological functions including cellular reproduction, segregation, and apoptosis [1]. Previous studies have been indicated that mirnas can act as either oncogenes or tumor suppressors [1 5]. Moreover, deregulation or dysfunction of mirnas may contribute to cancer development [6 9]. Nasopharyngeal carcinoma (NPC) is known as a nonlymphomatous, squamous cell malignancy, which is the cause of cancer-related mortality in many areas of the world. It is reported that NPC is the cause of 80,

2 000 new cases and 50,000 deaths every year [10]. Early diagnosis of NPC is difficult because the location of the cancer and the lack of clinical manifestation have crucial role in diagnosis of tumors. It is worth noting that NPC is strongly metastatic. Therefore, it can be beneficial to determine that important mechanisms contribute to progression and metastasis of NPC and identification of functional and clinical importance of a specific mirna may provide effective management of disease. It has been previously reported that mirnas could regulate cell cycle, apoptosis, invasion, and proliferation in NPC [11, 12], suggesting that it could play a key role in NPC tumorigenesis. Ectopic regulation of mir- 494 has been reported in different types of cancer including gastrointestinal, lung, and liver cancer, and brain tumor [13, 14]. On the other hand, upregulation of mir-21 has been reported in many kinds of cancer including renal, breast, gastric, and oral cancer [15 17]. It can be beneficial to clarify the role of mir-21 and mir-494 in NPC progression of NPC cancer. Therefore, we evaluated the expression level of mir-21 and mir- 494 in clinical NPC specimen and normal tissues. Furthermore, clinical relevance of mir-21 and mir-494 in NPC cases was investigated in the current study. Table 1 Association between mir-21 expression and clinicopathological features in NPC patients Characteristics Number of cases mir-21 expression P value Age (years) ± > ±1.47 Gender Male ± Female ±1.33 Degree of differentiation Undifferentiated ± Differentiated ±1.012 Histology Squamous ± Others ±1.13 Lymph node metastasis Positive ± Negative ±1.25 Distal metastasis Positive ± Negative ±1.18 TNM stage I II ± III IV ±1. 42 Table 2 Relationship between mir-494 expression and clinicopathological features in NPC patients Characteristics Number of cases mir-494 P value Mean age range (years) 1.31±1.014; ± > ±1.12 Gender Male ± Female ±1.30 Degree of differentiation Undifferentiated ± Differentiated ±1.22 Histology Squamous ± Others ±1.023 Lymph node metastasis Positive ± Negative ±1.46 Distal metastasis Positive ± Negative ±0.96 TNM stage I II ± III IV ±0.52 Materials and methods Samples Tumor Biol. A total of 34 samples were collected from patients with NPC who underwent surgery without chemotherapy or radiotherapy before the surgery at the different hospitals in Tehran, Tabriz, and Mashhad hospitals between March 2009 and December Furthermore, a total of 25 normal nasopharyngeal tissues were also collected in the present study. The specimens were stored in liquid nitrogen ( 80 C) until use. The Fig. 1 The relative expression level of mir-21 expression between NPC tissues and normal tissues

3 Fig. 2 The relative expression level of mir-494 between NPC tissues and normal tissues clinicopathological features are classified in Tables 1 and 2. This study was confirmed by the Ethics Committee, Iran Quantitative real-time PCR In present study, the total RNA was purified from NPC samples and normal control tissues using TRIzol reagent based on the constructor s service for user. Gene-specific primers were used to synthesize the mir-21 and mir-494, and U6 was applied as an internal specific cdnas (control) from the total RNA based on the TaqMan microrna assay. Furthermore, real-time PCR was carried out using an Invitrogen kit by system of Rotor-gene 6000 (Qiagen). Furthermore, expression rate of mirna was assessed utilizing the cycle threshold. The relative amount of mir-21 and mir-494 was normalized with respect to U6 RNA. Moreover, 2ΔΔCt method was used to calculate the fold-change among cancer and normal samples. The ΔΔCt (ΔΔCt=ΔCt tumor samples ΔCt control sample ) was used to qualify the expression rate of mir-21 and mir-494 in neoplastic and normal samples. Statistical analysis The differences of expression in neoplastic and normal tissues were evaluated using Student s t test. Moreover, Fig. 3 Correlation between mir- 21 and mir-494 expression and survival time in in NPC patients. Low expression was strongly associated with shorter survival compared with high expression (log-rank test, a P=0.002; b P=0.024) relationship between expression and clinicopathological factors was evaluated using two-sample Student s t test. Moreover, survival rates were analyzed using the logrank test and Kaplan-Meier method between neoplastic and normal tissue. Furthermore, we applied multivariate analysis using Cox proportional hazards model to assess clinicopathological factors linked to patient survival. Whole statistical analyses were implemented using SPSS software (V.16.0, USA). Differences were statistically significant at P<0.05. Results Our result indicated that mir-21 expression was higher in tumor tissue when compared with normal tissues (mean±sd 4.81±1.041; 2.13±0.87; P=0.035) (Fig. 1). Moreover, downregulation of mir-494 expression was detected in NPC compared with normal tissues (mean± SD 1.31±1.014; 4.02±1.081; P=0.023) (Fig. 2). In addition, NPC patients were divided into expression group (low and high group) based on the median expression level of mir-21 and mir-494. The result showed that 15 cases belonged to the low mir-21 expression group, while 19 cases were classified as high expression group. On the other hand, 22 cases were divided to the low rate of mir-494 expression, and 12 cases were determined to the great expression class in the present study. The results of the current study indicated that increased expression of mir-21 and decreased expression of mir-494 were strongly associated with high tumornode-metastasis (TNM) stage (P=0.023; P=0.016) and lymph node metastasis (P=0.031; P=0.041). Nevertheless, there was no significant relationship of mir-21 and mir-494 expression with other factors including age (P=0.621; P=0.561), sex (P=0.547; P=0.634), degree of differentiation (P=0.426; P=0.301), histology (P=0.321; P=0.432), and distal metastasis (P=0.582; P=0.472) (Tables 1 and 2).

4 Survival analysis Kaplan-Meier analyses and log-rank analysis were used to evaluate relationship of mir-21 and mir-494 expression level with survival in NPC patients. As shown in Fig. 3, shorter overall survival was significantly associated with high and low expression of mir-21 and mir-494 when compared with other group of patients that had low and high expression, respectively (log-rank test P=0.002; P=0.024). Multivariate Cox model indicated that high expression of mir-21, low expression of mir-494, and TNM stage were independently associated with poor survival of NPC patients (Tables 3 and 4). Discussion It can be important to understand the mechanism that contributes to the progression and metastasis of NPC. Moreover, determination of functional and clinical importance of a specific mirna may provide effective management of disease. It has been earlier announced that there is a correlation between mirna expressions and tumor prognosis [18, 19]. In the present study, mir-21 expression was upregulated in tumor tissue when compared with normal tissues. The upregulation of this MicroRNA has been previously reported in many kinds of malignancies including gastrointestinal cancer [5], lung cancer [20], prostate cancer [21], pancreatic cancer [22], hepatocellular cancer [23], breast cancer [24], malignant cancer [25], and NK-cell lymphoma [26]. The result of the present study is in agreement with the mentioned studies. On the other hand, downregulation of mir-494 expression was found in the current study, and ectopic regulation of mir-494 has been reported in different cancer types including gastrointestinal, lung, and liver cancer, and brain tumor [13, 14]. They indicated that mir-494 could contribute to occurrence and development of tumor. Shen Table 3 Multivariate analysis with a Cox proportional hazards model between mir-21 and clinicopathological factors Clinicopathological characteristics HR 95 % CI P value Sex Age TNM stage Degree of differentiation Histology Lymph node metastasis Distal metastasis mir-21 level Table 4 Multivariate analysis with a Cox proportional hazards model between mir-494 clinicopathological factors Clinicopathological characteristics HR 95 % CI P value SexCI Age TNM stage Degree of differentiation Histology Lymph node metastasis Distal metastasis mir-494 level et al. have reported that mir-494 was downregulated in prostate cancer and can suppress the proliferation and migration of prostate cancer cells by regulating CXCR4, which interfere in various cellular process and cancer progression [27]. Our result indicated that high expression of mir-21 and low expression of mir-494 can be related to tumor invasion and progression in NPC patients. In the current study, we investigated the association of two micrornas with pathological characteristics of NPC patients. The results suggested that high expression of mir-21 and low expression of mir-494 were strongly linked to high TNM stage and lymph node metastases that may be associated with tumor invasion and progression. Survival analysis and log-rank test revealed that patients with high expression of mir-21 had shorter overall survival than patients who had low mir-21 expression, indicating that mir-21 may be as a marker for poor prognosis in NPC patients. On the other hand, low level of mir-494 expression was associated with shorter time survival when compared with high expression group of patients, suggesting that mir-494 could play its role as a potential marker for prognosis of NPC. Multivariate Cox model indicated that high expression of mir-21, low expression of mir-494, and TNM stage were independently associated with poor survival of NPC patients. Further studies are needed to clarify the involved mechanism in mir-21 and mir-494 expression with progression of NPC. Conclusions In summary, our results indicated that upregulation of mir- 21was correlated with poor prognosis of NPC patients. On the other hand, downregulation of mir-494 may play a critical role as independent prognostic factor in NPC. Conflicts of interest None Tumor Biol.

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