MiR-210 has the capacity to serve as a diagnostic biomarker for laryngeal carcinoma patient

Early diagnosis represents a great challenge for laryngeal carcinoma patients. MiR-210 is involved in various human cancers. In this study, we aimed to investigate the diagnostic performance of serum miR-210 in laryngeal carcinoma. Methods In our study, qRT-PCR was performed to determine the serum miR-210 level in 137 laryngeal carcinoma patients and 79 healthy volunteers. The association of serum miR-210 level with clinical characteristics of the patients was estimated by chi-square test. ROC analysis was applied to evaluate the diagnostic value of miR-210 in laryngeal carcinoma.


Conclusion
MiR-210 serves as an oncogene in progression of laryngeal carcinoma. Serum miR-210 may be a potential diagnostic biomarker for laryngeal carcinoma.

Background
Laryngeal carcinoma is one of the most common cancers in head and neck region, with increasing morbidity and high mortality around the world [1]. Several risk factors are con rmed for laryngeal carcinoma, such as smoking, alcohol abuse, and exposure to carcinogens, however, the etiology of the cancer still remains unclear [2]. The cancer can be curable by surgical strategies or radiotherapy at early stages, but the prognosis of patients with advanced stages are far from satisfactory [3]. Tumor stage at initial diagnosis is a pivotal factor for outcomes of patients with laryngeal carcinoma [4]. Until now, early diagnosis of laryngeal carcinoma is mainly based on conventional biopsy which is frequently performed under local or general anesthesia [5]. In addition, CT, MRI, ultrasonography, and noninvasive imaging techniques are also used for early detection of the disease, but their diagnostic accuracy can not meet the clinical requirements [6][7][8]. Thus it is urgent to identify a novel and valuable biomarker which can help to achieve the early diagnosis for the laryngeal cancer patients.
MicroRNAs (miRNAs) are a class of short and non-coding RNAs with the length of 20-23 nucleotides [9].
Given their regulatory roles in gene expression at post-transcriptional level, miRNAs are involved in a variety of biological processes, including cell differentiation, proliferation, and apoptosis [10].
Dysregulation of miRNAs may contribute to occurrence and development of diseases, including cancers.
MiRNAs can function as oncogenes or tumor suppressors in tumorigenesis. The expression patterns of miRNAs show signi cant association with tumor initiation, development and progression, suggesting their potential as predictive biomarkers for human malignancies [11]. MiRNA-210 (MiR-210), a common member of miRNAs family, was reported to be involved in various human cancers, such as breast cancer, lung cancer, pancreatic carcinoma, etc [12][13][14]. However, the expression pro le of serum miR-210 and its clinical signi cance in laryngeal carcinoma were still poorly known.
In this study, we aimed to explore the expression pro le of serum miR-210 in laryngeal carcinoma, as well as its association with clinicopathologic characteristics. The potential diagnostic value of miR-210 in laryngeal carcinoma was also estimated in the current study.

Collection of patients and specimens
A total of 137 newly diagnosed laryngeal carcinoma patients were nally recruited in this study. Our study was approved by the ethics committee of Chinese PLA General Hospital. All the patients or their family signed informed consents in advance. In addition, 79 gender and age matched healthy volunteers were collected as the control group. In the control group, no one had been diagnosed with any malignancies.
The blood specimens were taken from all the participants on the morning in a collection tube with EDTA.
Then the samples were centrifugated at 3000 rpm for 10 min to isolate the serum sample. The supernate was stored at -80℃ until RNA extraction. The basic data of those patients with laryngeal cancer were recorded in Table 1, including age, sex, tumor size, subsite, histologic type, TNM stage, and distant metastasis. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted with the miRNeasy Mini Kit (Qiagen, Hilden Germany) following the manufacturer's instructions. The rst strand of cDNA was synthesized with the One Step PrimeScript miRNA cDNA Synthesis Kit (Takara Bio, Shiga, Japan). The relative expression level of miR-210 was measured using qRT-PCR method which was performed with a SYBR Premix Ex Taq™ kit (Takara, Dalian, China) on the Applied Biosystems 7900 Fast Real-Time PCR system (Applied Biosystems, Foster City, California, USA). U6 snRNA was used as an internal control, and the primers of miR-210 were forward: 5'-ACACTCCAGCTGGGCTGTGCGTGTGACAGCGG-3', reverse: 5'-CTCAACTGGTGTCGTGGA-3', and primers of U6 were forward: 5'-CTCGCTTCGGCAGCACA-3', reverse: 5'-AACGCTTCACGAATTTGCGT-3'. The relative expression level of miR-210 was normalized to that of U6 and calculated using 2 −ΔΔCt method. Each test was performed in triple.

Statistical analysis
In this study, all statistical analyses were performed with software of SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad, San Diego, CA, USA). The serum miR-210 expression level was expressed as mean ± SD, and student's t-tests was used to compare its differences between case and control groups. The relationship between serum miR-210 expression and various clinicopathological characteristics were assessed using Chi-square tests. To determine the diagnostic performance of serum miR-210 expression in laryngeal carcinoma, the receiver operating characteristic (ROC) curve was plotted based on the serum levels of miR-210 in healthy individuals and laryngeal cancer patients. P values < 0.05 were considered statistically signi cant.

Results
The overexpression of miR-210 in laryngeal cancer In order to investigate the expression pro le of miR-210 in 137 laryngeal cancer patients and 79 healthy volunteers, the qRT-PCR was performed. As shown in Fig. 1, the results suggested that miR-210 expression was signi cantly higher in laryngeal cancer tissues than that in healthy volunteers (P < 0.001).
Association between miR-210 and clinicopathological parameters of laryngeal cancer patients The patients were divided into high expression (n = 78) and low expression (n = 59) groups according to their mean expression value of miR-210. Chi-square tests was used to evaluate the effects of miR-210 expression on clinical characteristics of laryngeal carcinoma patients. Analysis results demonstrated that miR-210 expression was signi cantly associated with TNM stage (P = 0.000) and distant metastasis (P = 0.001). However there were no obvious relationship between the expression of miR-210 and the age, gender, tumor size, subsite or histologic type of patients with laryngeal carcinoma (all P > 0.05, Table 1).

Diagnostic value of miR-210 expression in laryngeal cancer patients
In our study, ROC curve was used to determine the accuracy of miR-210 in distinguishing laryngeal cancer patients and healthy group. The AUC value of the curve was 0.893, suggesting that the laryngeal carcinoma patients could be distinguished from the healthy group based on their serum levels of miR-210. The cut-off value of serum miR-210 for laryngeal carcinoma diagnosis was 4.685, with the sensitivity of 83.2% and the speci city of 84.8% (Fig. 2).

Discussion
Laryngeal carcinoma represents a frequently diagnosed head and neck cancer. Despite of the various available treatments, the clinical outcomes of the patients has not been signi cantly improved during the past three decades [15]. Low early diagnosis rate may be responsible for the high mortality [4]. Surgery and radiotherapy are effective treatments for patients diagnosed at early stages, but the therapeutic effects are limited for those diagnosed with advanced stages, due to the high recurrence rate [16]. Therefore, identi cation of novel biomarkers for early diagnosis may be a promising approach to improve the outcomes of the patients.
As gene expression regulators, miRNAs play important roles in various biological processes, such as development, cell proliferation, apoptosis, differentiation, as well as carcinogenesis [17,18]. Growing evidences have demonstrated that miRNAs as oncogenes or suppressors are involved in various human malignancies. Given their functional roles in tumor progression, miRNAs are considered as promising candidate biomarkers for cancer diagnosis, prognosis, and treatments. In laryngeal cancer, a variety of miRNAs biomarkers were identi ed. For examples, Wu er al. reported that laryngeal cancer tissues exhibited increased expression of miR-148a and miR-375 which might serve as diagnostic biomarkers for the cancer [19]. Zhang et al. reported that up-regulation of miR-23a in laryngeal cancer showed positive correlation with aggressive clinical parameters of the patients, moreover, its elevated expression predicted poor prognosis [20]. Based on the related studies, we speculated that the expression pro le of miRNAs showed signi cant association with tumor development and progression, and they held the potential to serve as predictive biomarkers for human malignancies.
MiR-210, a common member of miRNA family, has been determined to play an important role in tumourgenesis. The increased expression of miR-210 was observed in various cancer, such as lung adenocarcinoma, renal cell carcinoma, colorectal cancer, suggesting its carcinogenic function in these cancers [21][22][23]. In this study, we found that the expression level of miR-210 was higher in laryngeal carcinoma patients than that in healthy group. Moreover, the increased expression of miR-210 was signi cantly correlated with advanced TNM stage and positive distant metastasis. All the data revealed that miR-210 as a tumor oncogene played a promoting role in malignant development and progression of laryngeal carcinoma. This conclusion was consistent with the previous results obtained in other types of cancer.
Given its oncogenic roles in tumorigenesis, miR-210 was identi ed as a biomarker for several cancers. In clear cell renal cell carcinoma, up-regulation of miR-210 showed signi cant association with tumor recurrence and poor prognosis of the patients, suggesting its capacity as a prognostic indicator for the disease [24]. Wang et al. reported that serum level of miR-210 was signi cantly higher in colorectal cancer patients than that in the healthy individuals, moreover, its elevated expression was positively correlated with malignant tumor progression and poor prognosis. Circulating miR-210 might be a potential biomarker for early detection and prognosis evaluation of the disease [25]. In this study, we estimated the diagnostic performance of serum miR-210 in laryngeal cancer. The results suggested that miR-210 could distinguish the laryngeal cancer patients from the healthy individuals with high sensitivity and speci city. MiR-210 might be a potential diagnostic biomarker for laryngeal carcinoma patients. However, the sample size was relatively small, and the application value of serum miR-210 for laryngeal carcinoma required further identi cation. In addition, the carcinogenic mechanisms of miR-210 in laryngeal cancer were poorly known. Further analysis were still needed to address the related issues.

Conclusion
In conclusion, miR-210 is elevated in laryngeal carcinoma, and positively correlated with malignant tumor progression, Serum miR-210 may be a candidate biomarker for early detection of laryngeal carcinoma. The authors report no con icts of interest in this work.

Author Informatio
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. Figure 1 The relative expression of serum miR-210 in laryngeal cancer cases and healthy individuals. The expression of serum miR-210 was signi cantly increased in laryngeal cancer patients, compared to the