Expression of miRNAs in Papillary Thyroid Carcinomas Is Associated with BRAF Mutation and Clinicopathological Features in Chinese Patients

MicroRNAs (miRNAs) dysregulation has been shown to play a critical regulatory role in papillary thyroid carcinomas (PTCs). BRAF mutation is associated with poor clinicopathological outcomes in PTC. In order to identify a possible association between dysregulated miRNA expression and BRAF mutation as well as clinicopathological features in Chinese patients with PTC, we examined the expression levels of five reported dysregulated miRNAs (miRNA-221, miRNA-222, miRNA-146b, miRNA-181, and miRNA-21) and determined BRAF mutation status in 52 patients with PTC and 52 patients with benign thyroid nodules (BTNs). The expression levels of all five miRNAs were significantly increased in PTC when compared to BTN. The BRAF mutation occurred more frequently in PTC cases with advanced TNM stage. Importantly, miRNA-221, miRNA-222, miRNA-146b, and miRNA-181 expression levels were significantly higher in PTC patients with BRAF mutation. In addition, enhanced expression of miRNA-221 and miRNA-222 was found in patients with cervical lymph node metastasis and advanced TNM stage. Increased expression of miRNA-221 and miR-181 was evidenced in patients with larger tumors. These findings showed a potential role of this distinct profile of miRNAs in differentiating PTC from BTN. BRAF mutation might regulate or interact with miRNA in the pathogenesis and progression of PTC.


Introduction
Thyroid cancer is the most prevalent endocrine malignancy (accounting for >92% endocrine malignancy), which can be classified histologically into follicular epithelial cell-derived papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), anaplastic thyroid cancer (ATC), and parafollicular C-cell-derived medullary thyroid cancer (MTC) [1,2]. In the past several decades, the incidence of thyroid cancer worldwide has been steadily increasing, and this rising is mainly attributed to the increased diagnosis of PTC [2][3][4][5][6][7][8][9]. PTC is the most common histological type of thyroid cancers and accounts for approximately 80% of reported cases [10]. The overall prognosis of PTC patients is excellent after appropriate treatment. However, the mortality of PTC with aggressive clinicopathological features was much higher when compared with PTC without these clinicopathological features [1]. Therefore, it is of great importance to identify biomarkers that are associated with aggressive clinicopathological features of PTC, and these biomarkers might also serve as potential pharmacological targets for PTC.
BRAF mutation is the most common genetic alteration in thyroid malignances, exclusively existing in PTC and PTCderived ATC, but not in FTC and MTC. This mutation is found in about 45% of sporadic PTC. Numerous studies have consistently shown that BRAF mutation, which upregulates thyroid cell division and proliferation by activating MAPK pathway, is mutually exclusive with other common genetic alterations such as the rearrangement in transformation/papillary thyroid carcinoma (RET/PTC) signal and Ras 2 International Journal of Endocrinology mutation [11][12][13]. Importantly, many studies have proved that BRAF mutation is correlated with high-risk clinicopathological characteristics, such as larger tumor size, extrathyroidal invasion, local lymph node metastasis, distant metastasis, and advanced disease stages, suggesting that it is not only an independent oncogenic event for PTC tumorigenesis, but also involved in progression of PTC [14][15][16].
MicroRNAs (miRNAs) are small noncoding RNA molecules, about 21-25 nucleotides in length, which negatively regulate gene expression at the posttranscriptional level. More than 1000 miRNAs are found in humans and up to onethird of the total human mRNAs are predicted to be miRNA targets [17]. Aberrant miRNA expression has been described in a variety of tumors, including PTC [17][18][19][20][21]. He et al. reported that 17 miRNAs were upregulated in PTC compared to adjacent normal tissue and defined five miRNAs (miRNA-221, miRNA-222, miRNA-146b, miRNA-181, and miRNA-21) being able to predict cancer status [19]. Tetzlaff et al. revealed the upregulation of miRNA-21, miRNA-31, miRNA-221, and miRNA-222 by real-time RT-PCR in PTC compared to multinodular goiter [20]. Among the various studies reported, the expression of 5 miRNAs, including miRNA-221, miRNA-222, miRNA-146b, miRNA-181, and miRNA-21, has been shown to be dysregulated [22]. In our study, we sought to further validate the expression of these 5 previously reported miRNAs in differentiating PTC from benign thyroid nodules (BTNs) in Chinese patients. Moreover, the associations between miRNAs and BRAF mutation, as well as other clinicopathological features, will be addressed in the present study. Table 1), archival FFPE surgical samples previously diagnosed as PTC (52 samples) and BTN (52 samples), were obtained from the First Affiliated Hospital and Cancer Center of Sun Yat-sen University (Guangzhou, China). All samples of PTC characterized in this study were the classic variant. Samples of BTN included solitary nodule and multinodular goiter. Informed consents were provided by all subjects and the study protocol was approved by the ethics committee of Sun Yat-sen University. For RNA isolation, eight 10 m sections per sample were obtained from the selected block for extraction of miRNA with the miRNeasy FFPE kit (QIAGEN, Hilden, Germany). Genomic DNA was extracted from FFPE samples using the QIAamp DNA FFPE Tissue Kit (QIAGEN). The RNA and DNA yields were determined using the NanoDrop ND-1000 spectrophotometer (Nanodrop, USA).

BRAF Mutation and Clinicopathological Features of PTC.
When assessing the relationship between BRAF mutation and clinicopathological features, we found that BRAF mutation occurred more frequently in PTC patients (19/52) with advanced TNM stage ( = 0.014). However, other clinicopathological features, including tumor size and cervical lymph node metastasis, were not significantly different between patients with or without BRAF mutation.

Differences in miRNA Expression in PTC Patients with
Different Clinicopathological Features. We next accessed the differences in the expression levels of these 5 miRNAs in PTC patients with different clinicopathological characteristics ( Table 2). The levels of miRNA-221 and miRNA-181 were significantly higher in patients with tumor diameter ≥2 cm than <2 cm ( = 0.004 and = 0.008, resp.) (Figure 3(b)). The expression levels of miRNA-221 and miRNA-222 were significantly higher in PTCs with advanced tumor-nodemetastasis (TNM) stage ( = 0.004 and = 0.041, resp.) (Figure 3(c)). In addition, patients with lymph node metastasis had higher expression levels of miRNA-221 and miRNA-222 ( = 0.033 and = 0.014, resp.) (Figure 3(d)). There were no significant differences in these miRNA levels in PTC patients with other clinicopathological parameters, including age, gender, tumor multicentricity, heteromorphic nucleus, thyroid globulin quantification, and AMES (risk definition including age, metastases, extent, and size) score (Table 2).

Discussion
In the present study, we have reported the association of 5 previously reported miRNAs and BRAF mutation and the clinicopathological features in Chinese patients with PTC. We showed that the expression levels of these five miRNAs   The data are shown as median (range); value was determined by the Mann-Whitney U test. TNM: tumor node metastasis; LN: lymph nodes; AMES: age, metastasis to distant sites, extrathyroidal invasion, and tumor size scoring system.

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International Journal of Endocrinology (miRNA-221, miRNA-222, miRNA-146b, miRNA-181, and miRNA-21) were significantly higher in FFPE tissues from PTCs than BTNs. miRNA-221, miRNA-222, miRNA-146b, and miRNA-181 expression levels were significantly higher in PTC patients with BRAF mutation. Enhanced expression of miRNA-221 and miRNA-222 was found in patients with cervical lymph node metastasis and advanced TNM stage. Increased expression of miRNA-221 and miR-181 was evidenced in patients with larger tumors. Thyroid nodule is a common entity in the clinic. Although most of the thyroid nodules are benign, 5% to 30% of nodules are still malignant and require surgical intervention [25]. At present, pathological study is the golden criteria of diagnosis of thyroid nodules. However, there are several limitations in the pathological diagnosis of thyroid nodules, including inadequate sampling, indeterminate result, and possible misdiagnosis [26]. During the past decades, gene detection techniques have developed considerably, which make it possible to further explore tumorigenesis and progression of PTC at molecular level and to discover potential diagnostic and prognostic biomarkers. Among these techniques, detection of some specific miRNA expression levels is a promising method to provide information for molecular diagnosis of PTC [18][19][20]. In our study on utility of miRNA profiling, we have correlated this data with pathology of the surgical specimen, a gold standard of diagnosis, and we believe that our findings are certainly helpful in providing potentially diagnostic biomarker for PTC before operation. Our results demonstrated that each of these 5 miRNAs or the 5-miRNA set (miRNA-221, miRNA-222, miRNA-146b, miRNA-181, and miRNA-21) could be used as a potential biomarker to differentiate PTC from BTN, with highest diagnostic value for miRNA-146b (sensitivity of 90.4% and specificity of 88.5% at an AUC of 0.952) and 5-miRNA set (sensitivity of 90.4% and specificity of 88.5% at an AUC of 0.937). Consider that the upregulation level of miRNA-146b is not consistently reported [22]. Therefore, we suggest that use of the 5-miRNA set as a potential diagnostic biomarker for PTC might be more helpful. In order to further validate the potential diagnostic value of miRNA in the preoperative evaluation of thyroid nodules, we will analyze the proposed miRNAs data in "intermediate" or "suspicious" FNA cases based on current results in our future prospective study.
BRAF mutation is the most common mutation in PTC, which leads to a constitutive activation of the MAPK pathway. Activation of this pathway is a common and important mechanism in genesis and progression of human cancers through upregulating cell division and proliferation [11][12][13]. A number of studies have reported that BRAF mutation was associated with aggressive PTC characteristics, such as extrathyroidal extension, lymph node metastasis, tumor recurrence, decreased survival, and need for cervical reoperation [14][15][16]. In our present study, we found that BRAF mutation occurred more frequently in the PTC patients with advanced TNM stage, which is consistent with previous studies [14,27,28].
The relationship between BRAF mutation and miRNA in PTC had been investigated. However, discrepant results were found from different studies. Chou et al. demonstrated that miRNA-146b was overexpressed in PTC with BRAF mutation [29]. Yip et al. also documented that overexpression of miRNA-146b was associated with aggressive behavior in BRAF mutant PTC [30]. However, Sheu et al. reported that there was no correlation between BRAF mutation and the expression profile of a set of miRNAs (miRNA-221, miRNA-222, miRNA-146b, miRNA-181, and miRNA-21) in PTC [31]. In our study, miRNA-221, miRNA-222, miRNA-146b and miRNA-181 expression levels were significantly higher in PTC patients with BRAF mutation. The discrepancy between these studies could be due to different sample origins and the fundamental methodological differences used in the profiling studies. Therefore, it is of interest to further elucidate how these miRNAs and BRAF mutation interact with each other in the pathogenesis and progression of PTCs.
miRNA-221 and miRNA-222, which are encoded in tandem on the X chromosome, have been shown to be overexpressed in numerous human tumors including PTC [32][33][34][35]. It was reported that p27/kip1 and p57/kip2 were functional targets of miRNA-221 and miRNA-222. Over-expression of miRNA-221 and miRNA-222 in cancer cell lines facilitates cell proliferation via downregulation of the expression of p27/kip1 and/or p57/kip2, both of which are cell cycle inhibitors and tumor suppressors [35]. He et al. have demonstrated the upregulation of miRNA-221 in uninvolved thyroid tissue from individuals with PTC compared to normal thyroid tissue from individuals without thyroid disease [19]. Tetzlaff et al. confirmed that not only miRNA-221 but also miRNA-222 was overexpressed in some cases of multinodular goiter in comparison with PTC [20], suggesting that upregulation of miRNA-221 and miRNA-221 is perhaps early events in thyroid carcinogenesis. Nevertheless, our current study showed that expression of miRNA-221 and miRNA-222 is significantly upregulated in PTC compared to BTN and in patients with advanced TNM stage and lymph node metastasis. Similar to our findings, a report from Taiwan showed that miRNA-221 and miRNA-222 were significantly associated with extrathyroidal invasion of PTC [29]. Taken together, upregulation of miRNA-221 and miRNA-222 may not only be involved in the development of PTC, but also in invasion and metastasis of PTC.
The roles of miR-181 in human carcinogenesis have been explored. miR-181 has been demonstrated to function both as oncogene and tumor suppressor depending on the origin of tissues and cells. Accordingly, miR-181 overexpression has been observed in breast [36] and colon tumors [37]. The downregulation of this miRNA has also been noticed in gliomas [38] and aggressive CLL [39]. In our study, miR-181 expression was significantly higher in PTC versus BTN, suggesting that miR-181 might serve as an oncogene. Moreover, the expression level of miRNA-181 was higher in PTC with larger tumor size. Meng et al. reported that miR-181, by directly targeting on RASSF1A, TIMP3, and NLK, might be involved in hepatocellular cancer stem cells differentiation and invasion [40]. Considering the roles of miR-181 in carcinogenesis, the target genes of miRNA-181 in PTC are required to be identified in the future.
miRNA-146b over-expression has been shown in the majority of PTC studies [19,20,41]. In a series composing of seven pathologically distinct thyroid nodules, Chen et al. demonstrated that miRNA-146b, but not miRNA-221 and miRNA-222, was most consistently overexpressed in PTC [41]. Chou et al. suggested that miR-146b is a novel prognostic factor of PTC, and overexpression of miR-146b significantly increased cell migration and invasiveness [42]. In our study, we showed that miRNA-146b might be more helpful for distinguishing patients with PTC from BTN subjects, while there was no significant difference in miRNA-146b expression in PTC patients with different clinicopathological features. As to the expression levels of miRNA-21, although it was significantly higher in PTC versus BTN, there was substantial expression overlap between the two lesions and there was also no significant difference in miRNA-21 level in PTC patients with any tested clinicopathological features. These miRNAs might act as a tumor-related miRNA, but their roles in the pathogenesis of PTC still need further investigation.
In conclusion, we have confirmed that 5 previously reported miRNAs (miRNA-221, miRNA-222, miRNA-146b, miRNA-181, and miRNA-21) were up-regulated in Chinese patients with PTC. Enhanced expression of these miRNAs occurred more frequently in PTC patients with BRAF mutation and some clinicopathological features. Our findings showed a role of selected miRNAs as potential biomarkers in differentiating PTC from BTN and as a prognostic indicator of PTC in Chinese population.