Abstract
Objective
Alterations in microRNA (miRNA) expression have been described in thyroid tumors, suggesting a role for miRNAs in thyroid carcinogenesis. BRAFV600E is the most frequently identified genetic alteration in papillary thyroid carcinoma (PTC). We investigated the link between BRAFV600E status and the expression of miRNAs in PTC and analyzed the associations of these factors with clinicopathological characteristics.
Design and methods
Prospective study of patients who underwent thyroid surgery between October 8, 2008 and November 1, 2010. BRAFV600E status was determined by mutant allele-specific amplification PCR and direct sequencing of exon 15 of the BRAF gene in 69 PTC tissues and 69 respective paracancerous normal thyroid tissues. Initially, miRNA expression was analyzed in 12 PTC tissues and three associated paracancerous tissues using a miRNA microarray. miRNAs differentially expressed between BRAFV600E-positive and -negative PTC tissues were then validated by real-time quantitative PCR on 69 PTC tissues and 69 paracancerous tissues. We also explored the associations between BRAFV600E status or differential miRNA expression and clinicopathological characteristics.
Results
The mutation rate of BRAFV600E in PTC was 47.8%. Twelve miRNAs were upregulated and six were downregulated in PTC tissues, among which miR-15a, 15a*, 34a*, 34b*, 551b, 873, 876-3p, and 1274a were first identified. miR-21* and 203 were significantly dysregulated (P<0.05) in PTC tissues with BRAFV600E. Additionally, there were significant associations (P<0.05) between BRAFV600E and a higher tumor–node–metastasis staging (III/IV), and between miR-21* over-expression and lymph node metastasis.
Conclusions
We identified two miRNAs that are differentially expressed in PTC tissues with BRAFV600E and revealed their associations with clinicopathological features. These findings may lead to the development of a potential diagnostic biomarker or prognostic indicator of PTC.
Introduction
Thyroid carcinoma is one of the most common endocrine malignancies, accounting for ∼1% of all newly diagnosed cancers (1). It consists of four major histological types – papillary thyroid carcinoma (PTC), follicular thyroid carcinoma, medullary thyroid carcinoma, and anaplastic thyroid carcinoma – of which PTC accounts for more than 80% (2). The biological behavior and growth patterns of PTC are variable: most have a low potential for malignancy and a favorable prognosis, while others present with clinicopathological characteristics associated with a poor prognosis; thesecharacteristics include an older onset age, a larger tumor diameter, extrathyroidal invasion, distant metastases,and aggressive histological variants (3, 4). Surgery (sometimes with adjuvant radiotherapy) is still the most effective approach for patients with PTC.
Previous studies (1, 5) have demonstrated genetic alterations in PTC: RET gene rearrangements (referred to as RET/PTC), NTRK1 gene rearrangements (referred to as TRK), and RAS or BRAF gene mutations, which are collectively involved in the RET/PTC (TRK)–RAS–BRAF–MEK–MAPK signaling pathway. V600E in BRAF, a thymine-to-adenine transversion at nucleotide 1799, is a hot spot for mutations in patients with PTC. It affects exon 15 of the BRAF gene and is present in 29–83% of PTC cases (6, 7, 8, 9). Nucera et al. (10) revealed that BRAFV600E is required to maintain and promote thyroid cancer progression. Some other studies (8, 11, 12, 13, 14) have indicated that BRAFV600E is associated with an increased incidence of poor clinicopathological parameters in PTC.
MicroRNAs (miRNAs), short RNAs that are usually 21–25 nucleotides in length and are generated from single-stranded miRNA gene transcripts, represent a recently identified class of endogenous noncoding RNAs. Their regulatory influence is mediated by their interactions with a protein complex called the RNA-induced silencing complex, which delivers them to their mRNA targets (15). Previous studies (16, 17, 18, 19) have demonstrated that miRNAs play a role not only in individual growth and development but also in regulating the expression of well-known oncogenes and tumor suppressor genes, and increasingly, dysregulated miRNAs have been identified in various malignancies. Recently, using miRNA microarrays and miRNA real-time quantitative PCR (qPCR), several studies (18, 19, 20, 21, 22, 23, 24, 25, 26) have reported dysregulated miRNAs (such as miR-7, 21, 30, 31, 34a, 144, 146, 146b, 155, 181b, 187, 200, 200b, 221, and 222) in PTC, suggesting that these are promising targets for studying the genesis and development of PTC.
The association between BRAFV600E status and miRNA expression in PTC has not been thoroughly examined. Cahill et al. (27) and Nikiforova et al. (18) showed dysregulated miRNAs (miR-127, 130a, 141, 144, 146b, 155, 187, 200a, 200b, 221, and 222) in thyroid-derived cell lines and tissues with BRAFV600E respectively. Additionally, resulting from the rapid development of miRNA microarray, more and more human miRNAs have been identified. In this study, we i) compared miRNA expression between PTC tissues and paracancerous normal thyroid tissues using an up-to-date miRNA microarray and determined the differentially expressed miRNA in PTC tissues with BRAFV600E and ii) explored the associations between BRAFV600E status or differential miRNA expression and the clinicopathological characteristics of PTC.
Materials and methods
This study complies with the Declaration of Helsinki and was approved by the Local Ethics Committee. Informed consent was obtained from each subject or his/her guardian.
Selection of cases and controls
The current study was conducted on patients of Asian descent who had undergone thyroid surgery between October 8, 2008 and November 1, 2010. All patients were from southeast China and were admitted to the Thyroid Department of The First Affiliated Hospital of Sun Yat-Sen University. Lesions and paracancerous thyroid tissues (the latter ones were resected from the margin of the post-resection samples and confirmed by both naked eye and hematoxylin and eosin staining to contain no apparent abnormal tissues) were successfully obtained from each of 69 patients with PTC (confirmed by hematoxylin and eosin staining, according to the widely accepted histological diagnostic criteria (28)). All the tissues were collected from surgically resected tissues and stored immediately at −80 °C until analysis. The 69 patients (male:female=1:4.3) were aged 14–75 years (median, 41 years; interquartile range, 23 years); 59 had classical PTC while the other 10 had the follicular variant of PTC. The tumor size was 2.48±1.31 cm (mean±s.d.); 31 cases (44.9%) were solitary lesions, 36 cases (52.2%) presented with lymph node metastasis, 25 cases (36.2%) presented with extrathyroidal invasion, and 48 cases (69.6%) were tumor–node–metastasis (TNM) staging (29) I/II.
RNA and DNA extraction
Genomic DNA and RNA were extracted from frozen specimens by standard techniques. Total RNA and DNA were extracted using TRIzol reagents (Invitrogen) and a TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) respectively according to the manufacturer's instructions. The RNA and DNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Only RNA samples that showed intact 18S and 28S ribosomal bands upon 1% agarose gel electrophoresis, and for which the A260/A280 ratio was 1.9–2.1 and the 28S/18S ratio was ≥2.0, were deemed acceptable.
Detection of BRAFV600E
Mutant allele-specific amplification PCR was conducted essentially as described previously (30) to detect BRAFV600Emutation in the 69 PTC tissues and the 69 paracancerous thyroid tissues. KTC-1 (a thyroid carcinoma cell line with BRAFV600E) and WRO (a thyroid carcinoma cell line without BRAFV600E) were used as positive and negative controls respectively. PCR products were analyzed by 2% agarose gel electrophoresis. At the same time, according to a previous study (30), exon 15 of the BRAF gene was PCR amplified from genomic DNA from the 69 PTC tissues and the 69 paracancerous thyroid tissues. PCR products were sequenced by Invitrogen. The data were analyzed using Lasergene Software (version 7.1.0; DNASTAR, Madison, WI, USA), comparing the sequences with the wild type human sequence obtained from the National Center for Biotechnology Information Gene database (http://www.ncbi.nlm.nih.gov/gene/673). PCR primers were synthesized by Invitrogen China.
miRNA microarray analysis
miRNA expression was analyzed using a Human miRNA Microarray System Version 3 (Agilent Technologies) according to the manufacturer's instructions, with probe sets for 866 human miRNAs and 89 viral miRNAs (Sanger miRBase release 12.0). In brief, using a miRNA Complete Labeling and Hyb Kit (Agilent Technologies), 120 ng total RNA was fluorescently labeled with Cy3 in a hybridization oven (Agilent Technologies) for 20 h at 55 °C, with rotation at 20 rounds per minute. Microarray hybridization, data generation, and normalization were performed by the Shanghai Biochip Corporation (Shanghai, China), following standard Agilent protocols. MeV 4.7.4 (http://www.tm4.org/mev/) was used for bioinformatic analysis and visualization of microarray data.
miRNA qPCR
cDNA was synthesized from 10 ng total RNA and extracted from 69 PTC tissues and 69 associated paracancerous thyroid tissues, using the TaqMan miRNA RT Kit (Applied Biosystems, Inc.). qPCR was performed to assess the expression of differentially expressed miRNAs, which were identified in BRAFV600E-positive PTC tissues compared with BRAFV600E-negative PTC tissues by the miRNA microarray. The expression levels of miRNAs were quantified using the miRNA-specific TaqMan miRNA Assay Kit (Applied Biosystems). According to the manufacturer's instructions, qPCR was performed on the Applied Biosystems 7500 Sequence Detection system. The expression of miRNAs was defined based on the threshold of amplification cycle number (Ct), and the quantity of each miRNA was normalized to the expression of U6 small nuclear RNA using the equation of 2−ΔCt (ΔCt=CtmiRNA−CtU6).
Statistical analysis
The miRNA microarray data were analyzed using Significance Analysis of Microarrays (SAM) version 3.0 (http://www-stat.stanford.edu/∼tibs/SAM). miRNAs were considered to be significantly differentially expressed only under the condition that the false discovery rate=0. Other data were analyzed using SPSS version 17.0 (SPSS, Inc.); P<0.05 was considered statistically significant. For the qPCR data, the Wilcoxon rank sum test was used to compare PTC tissues with and without BRAFV600E, and the paired t-test was used to compare BRAFV600E-positive PTC tissues with their associated paracancerous thyroid tissues. Moreover, the χ2 test (for age at diagnosis, gender, number of lesions, lymph node metastasis, TNM staging, and metastasis or recurrence) and the Wilcoxon rank sum test (for tumor size) were also used.
Results
miRNA expression profiles
Initially, miRNA expression was analyzed in 12 continuously PTC tissues plus paracancerous thyroid tissues from three out of 12 PTC patients. Figure 1 shows 12 upregulated miRNAs (fold-change >2) and six downregulated miRNAs (fold-change <0.5 for all except miR-30a) in PTC tissues, with statistical differences (P<0.05).
Heat maps of miRNA expression in papillary thyroid carcinomas and normal thyroid tissues by SAM analysis. Green bars indicate low expression and red bars indicate high expression. Nos 1–3 are normal thyroid tissues adjacent to the papillary thyroid carcinomas of Nos 7, 8, and 12 respectively. Twelve significantly upregulated (P<0.05) miRNAs (A) and six significantly downregulated (P<0.05) miRNAs (B) were found in 12 papillary thyroid carcinomas (Nos 4–15), compared with those expressed in three normal thyroid tissues (Nos 1–3). Full colour version of this figure available via http://dx.doi.org/10.1530/EJE-12-1029.
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
Heat maps of miRNA expression in papillary thyroid carcinomas and normal thyroid tissues by SAM analysis. Green bars indicate low expression and red bars indicate high expression. Nos 1–3 are normal thyroid tissues adjacent to the papillary thyroid carcinomas of Nos 7, 8, and 12 respectively. Twelve significantly upregulated (P<0.05) miRNAs (A) and six significantly downregulated (P<0.05) miRNAs (B) were found in 12 papillary thyroid carcinomas (Nos 4–15), compared with those expressed in three normal thyroid tissues (Nos 1–3). Full colour version of this figure available via http://dx.doi.org/10.1530/EJE-12-1029.
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
Heat maps of miRNA expression in papillary thyroid carcinomas and normal thyroid tissues by SAM analysis. Green bars indicate low expression and red bars indicate high expression. Nos 1–3 are normal thyroid tissues adjacent to the papillary thyroid carcinomas of Nos 7, 8, and 12 respectively. Twelve significantly upregulated (P<0.05) miRNAs (A) and six significantly downregulated (P<0.05) miRNAs (B) were found in 12 papillary thyroid carcinomas (Nos 4–15), compared with those expressed in three normal thyroid tissues (Nos 1–3). Full colour version of this figure available via http://dx.doi.org/10.1530/EJE-12-1029.
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
Detection of BRAFV600E
Combining mutant allele-specific amplification PCR and sequencing of BRAF exon 15, we determined that BRAFV600E was present in 47.8% (33/69) of 69 PTC tissues (Fig. 2) and was absent from all 69 paracancerous thyroid tissues.
Detection of BRAFV600E in papillary thyroid carcinomas by DNA sequencing and mutant allele-specific amplification PCR. (A) A BRAFV600E-negative DNA sample shows a single peak at nucleotide 1799 (arrow); (B) a BRAFV600E-positive (1799 T>A) DNA sample shows a double peak at nucleotide 1799 (arrow); (C) arrow shows the PCR products obtained by primers specific for mutant; (D) arrow shows the PCR products obtained by primers specific for wild type. M, marker (100–600 bp); P, positive control KTC-1 cell line (a thyroid carcinoma cell line with BRAFV600E); N, negative control MRO cell line (a thyroid carcinoma cell line without BRAFV600E). Full colour version of this figure available via http://dx.doi.org/10.1530/EJE-12-1029.
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
Detection of BRAFV600E in papillary thyroid carcinomas by DNA sequencing and mutant allele-specific amplification PCR. (A) A BRAFV600E-negative DNA sample shows a single peak at nucleotide 1799 (arrow); (B) a BRAFV600E-positive (1799 T>A) DNA sample shows a double peak at nucleotide 1799 (arrow); (C) arrow shows the PCR products obtained by primers specific for mutant; (D) arrow shows the PCR products obtained by primers specific for wild type. M, marker (100–600 bp); P, positive control KTC-1 cell line (a thyroid carcinoma cell line with BRAFV600E); N, negative control MRO cell line (a thyroid carcinoma cell line without BRAFV600E). Full colour version of this figure available via http://dx.doi.org/10.1530/EJE-12-1029.
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
Detection of BRAFV600E in papillary thyroid carcinomas by DNA sequencing and mutant allele-specific amplification PCR. (A) A BRAFV600E-negative DNA sample shows a single peak at nucleotide 1799 (arrow); (B) a BRAFV600E-positive (1799 T>A) DNA sample shows a double peak at nucleotide 1799 (arrow); (C) arrow shows the PCR products obtained by primers specific for mutant; (D) arrow shows the PCR products obtained by primers specific for wild type. M, marker (100–600 bp); P, positive control KTC-1 cell line (a thyroid carcinoma cell line with BRAFV600E); N, negative control MRO cell line (a thyroid carcinoma cell line without BRAFV600E). Full colour version of this figure available via http://dx.doi.org/10.1530/EJE-12-1029.
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
Expression of miRNAs relative to BRAFV600E status
Two miRNAs (miR-21* and 203) were upregulated in seven BRAFV600E-positive PTC tissues compared with five BRAFV600E-negative PTC tissues (Fig. 3A), with statistical differences (P<0.05). These findings were confirmed by qPCR (Fig. 3B and C). To further validate the association between BRAFV600E and these two miRNAs, the expressions of miR-21* and 203 were tested by qPCR on the other 57 PTC tissues and all 33 paracancerous thyroid tissues from BRAFV600E-positive PTC patients. miR-21* and 203 were upregulated in 45 and 41 PTC tissues respectively. And both of them were significantly dysregulated (P<0.05) in BRAFV600E-positive PTC tissues compared with BRAFV600E-negative PTC tissues or the associated paracancerous thyroid tissues, with coherence of changing trend (Fig. 4).
Differential expressions of miR-21* and 203 between BRAFV600E-positive and -negative papillary thyroid carcinomas (PTC). (A, B and C) Expressions of miR-21* and 203 in five BRAFV600E-negative (wild type, Nos 11–15) PTC tissues and seven BRAFV600E-positive (mutant type, Nos 4–10) PTC tissues using miRNA microarray (A) and real-time quantitative PCR (B and C). Green bars indicate low expression and red bars indicate high expression. Full colour version of this figure available via http://dx.doi.org/10.1530/EJE-12-1029.
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
Differential expressions of miR-21* and 203 between BRAFV600E-positive and -negative papillary thyroid carcinomas (PTC). (A, B and C) Expressions of miR-21* and 203 in five BRAFV600E-negative (wild type, Nos 11–15) PTC tissues and seven BRAFV600E-positive (mutant type, Nos 4–10) PTC tissues using miRNA microarray (A) and real-time quantitative PCR (B and C). Green bars indicate low expression and red bars indicate high expression. Full colour version of this figure available via http://dx.doi.org/10.1530/EJE-12-1029.
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
Differential expressions of miR-21* and 203 between BRAFV600E-positive and -negative papillary thyroid carcinomas (PTC). (A, B and C) Expressions of miR-21* and 203 in five BRAFV600E-negative (wild type, Nos 11–15) PTC tissues and seven BRAFV600E-positive (mutant type, Nos 4–10) PTC tissues using miRNA microarray (A) and real-time quantitative PCR (B and C). Green bars indicate low expression and red bars indicate high expression. Full colour version of this figure available via http://dx.doi.org/10.1530/EJE-12-1029.
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
miR-21* and 203 expressions in papillary thyroid carcinomas (PTC) and paracancerous normal thyroid tissues by real-time quantitative PCR. (A and B) The expressions of miR-21* and 203 in 36 BRAFV600E-negative (wild type) PTC tissues and in 33 BRAFV600E-positive (mutant type) PTC tissues; (C and D) the expressions of miR-21* and 203 in 33 BRAFV600E-positive PTC tissues and in 33 associated paracancerous normal thyroid tissues (normal).
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
miR-21* and 203 expressions in papillary thyroid carcinomas (PTC) and paracancerous normal thyroid tissues by real-time quantitative PCR. (A and B) The expressions of miR-21* and 203 in 36 BRAFV600E-negative (wild type) PTC tissues and in 33 BRAFV600E-positive (mutant type) PTC tissues; (C and D) the expressions of miR-21* and 203 in 33 BRAFV600E-positive PTC tissues and in 33 associated paracancerous normal thyroid tissues (normal).
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
miR-21* and 203 expressions in papillary thyroid carcinomas (PTC) and paracancerous normal thyroid tissues by real-time quantitative PCR. (A and B) The expressions of miR-21* and 203 in 36 BRAFV600E-negative (wild type) PTC tissues and in 33 BRAFV600E-positive (mutant type) PTC tissues; (C and D) the expressions of miR-21* and 203 in 33 BRAFV600E-positive PTC tissues and in 33 associated paracancerous normal thyroid tissues (normal).
Citation: European Journal of Endocrinology 168, 5; 10.1530/EJE-12-1029
Relationships between BRAFV600E status or differential miRNA expression and the clinicopathological characteristics
Significant associations (P<0.05) were identified for the 69 PTC patients between BRAFV600E positivity and a higher TNM stage (III/IV), and between miR-21* over-expression and lymph node metastasis (Table 1). By contrast, there was no significant association (P>0.05) between miR-203 expression and the clinicopathological characteristics (Table 1).
Relationships between clinicopathological characteristics and BRAFV600E status, miR-21* expression, or miR-203 expression in patients with papillary thyroid carcinoma (n=69).
| BRAFV600E status | miR-21* | miR-203 | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Parameter | Mutant type (%; n1=33, n2=31) | Wild type (%; n1=36, n2=35) | P | Upa (%; n1=45, n2=43) | Downb (%; n1=24, n2=23) | P | Upa (%; n1=41, n2=39) | Downb (%; n1=28, n2=27) | P |
| Age | 0.124 | 0.408 | 0.436 | ||||||
| ≥45 years | 18 (55) | 13 (36) | 23 (51) | 10 (42) | 20 (49) | 11 (39) | |||
| <45 years | 15 (45) | 23 (64) | 22 (49) | 14 (58) | 21 (51) | 17 (61) | |||
| Gender | 0.828 | 0.935 | 0.863 | ||||||
| Male | 6 (18) | 7 (19) | 9 (20) | 4 (17) | 8 (20) | 5 (18) | |||
| Female | 27 (82) | 29 (81) | 36 (80) | 20 (83) | 33 (80) | 23 (82) | |||
| Number of lesions | 0.570 | 0.260 | 0.837 | ||||||
| Single | 16 (48) | 15 (42) | 18 (40) | 13 (54) | 20 (49) | 11 (39) | |||
| Multiple | 17 (52) | 21 (58) | 27 (60) | 11 (46) | 21 (51) | 17 (61) | |||
| Lymph node | 0.390 | 0.005 | 0.765 | ||||||
| N0 | 14 (42) | 19 (53) | 16 (36) | 17 (71) | 19 (46) | 14 (50) | |||
| N1 | 19 (58) | 17 (47) | 29 (64) | 7 (29) | 22 (54) | 14 (50) | |||
| TNM stagingc | 0.043 | 0.715 | 0.941 | ||||||
| I and II | 17 (52) | 27 (75) | 28 (62) | 16 (67) | 26 (63) | 18 (64) | |||
| III and IV | 16 (48) | 9 (25) | 17 (38) | 8 (33) | 15 (37) | 10 (36) | |||
| Metastasis or recurrenced | 0.597 | 1.000 | 1.000 | ||||||
| Yes | 2 (6) | 1 (3) | 2 (5) | 1 (4) | 2 (5) | 1 (4) | |||
| No | 29 (94) | 34 (97) | 41 (95) | 22 (96) | 37 (95) | 26 (96) | |||
n1 is the total number of patients available for the data of age at diagnosis, gender, number of lesion, and tumor–node–metastasis (TNM) staging. n2 is the total number of patients available for the data of metastasis or recurrence by 20–45 months of follow-up (median, 32 months; interquartile range, 11 months).
Expression of the miRNA in tumor tissue was higher than that in associated paracancerous normal thyroid tissue.
Expression of the miRNA in tumor tissue was lower than that in associated paracancerous normal thyroid tissue.
TNM classification is defined according to the American Joint Committee on Cancer (2010).
Confirmed by color Doppler ultrasonography or computed tomography during the follow-up period.
Discussion
Although PTC is the most common type of thyroid carcinoma, and also has the best prognosis, local recurrence or distant metastasis can occur (3, 4). Moreover, some studies (8, 11, 12, 13) have pointed out the increased incidence of poor clinicopathological features in PTC with BRAFV600E. Owing to the great progress in the field of miRNA, it has become a hot research topic in kinds of diseases (including PTC). Limited in PTC and highlighting the paracancerous normal thyroid tissues as control group, the current study provided new insights into the following by up-to-date miRNA microarray and BRAFV600E detection: i) besides some previously reported (18, 19, 20, 21, 22, 23, 24, 25, 26) miRNAs, a set of miRNAs were dysregulated in PTC; ii) the expressions of miR-21* and 203 were significantly associated (P<0.05) with BRAFV600E status; and iii) over-expression of miR-21* was significantly associated (P<0.05) with poor clinicopathological prognostic feature.
According to recent reports (31, 32, 33), the differential expression of miRNAs in different thyroid nodules, sampled by fine-needle aspiration, may improve the preciseness of preoperative diagnosis. As early as 2005, He et al. (20) demonstrated that three miRNAs (miR-146, 221, and 222) were significantly upregulated in PTC, using a miRNA microarray. Similar studies (18, 19, 21, 22, 23, 24, 25, 26) have appeared in the later literature. Because of the rapid development of miRNA microarray technology (there are now more than 1000 identified human miRNAs), using up-to-date miRNA microarray may help to enrich the knowledge about miRNA expression in PTC. In the current study, we found 18 dysregulated miRNAs, among which miR-15a, 15a*, 34a*, 34b*, 551b, 873, 876-3p, and 1274a were first found in PTC.
BRAFV600E, formerly named BRAFV599E, was first discovered by Davies et al. (34) in malignant melanoma. It is believed that in thyroid tissues (including normal tissue, benign tumor tissue, and malignant tumor tissue), BRAFV600E is restricted to PTC and poorly differentiated or anaplastic carcinomas arising from PTC (30, 35, 36). We detected BRAFV600E in nearly half of the PTC tissues but in none of the associated paracancerous thyroid tissues, implying that it was a somatic mutation. There is a wide range of reported (6, 7, 8, 9) BRAFV600E mutation rates, from 29 to 83%; this might be attributable to ethnic heterogeneity and different sample sizes.
Few studies have examined the association between BRAFV600E and miRNA expression in PTC. On the one hand, BRAFV600E has an effect on activating BRAF–MEK–MAPK signaling pathway (37); on the other hand, miRNAs are posttranscriptional regulators that bind to complementary sequences on target mRNAs, usually resulting in target degradation, translational repression, or gene silencing (15). Cahill et al. (27) investigated the expression of 160 miRNAs in a normal thyroid cell line and two thyroid cell lines containing BRAFV600E; three miRNAs (miR-141, 200a, and 200b) were significantly upregulated and three (miR-127, 130a, and 144) were significantly downregulated in the mutation-carrying cell lines. Nikiforova et al. (18) selected six out of the 158 most highly expressed human miRNAs in PTC tissue and further explored their expressions in 23 PTC tissues (with/without BRAF or RAS gene mutations, or RET/PTC), revealing that five miRNAs (miR-146b, 155, 187, 221, and 222) were differentially expressed among the groups. Sheu et al. (38) assessed the expressions of five selected miRNAs (miR-21, 146b, 181b, 221, and 222) in 56 PTC tissues and concluded that they were not associated with BRAFV600E status. In our study, we initially identified two differentially expressed miRNAs (miR-21* and 203) between 12 BRAFV600E-positive and -negative PTC tissues by both miRNA microarray and qPCR. This was further validated by qPCR in a total of 69 PTC tissues and 33 paracancerous thyroid tissues from BRAFV600E-positive PTC patients. Previous studies have revealed that: activator protein 1 (AP-1) activated the miR-21 transcription in a human promyelocytic cell line (39) and increased the expression of miR-203 in human keratinocytes (40); MAPK signaling pathway had an effect on activating AP-1 (41); and the effect of BRAFV600E was mediated by the BRAF–MAPK signaling pathway (37). Taken together, we hypothesize that the relationship between BRAFV600E and over-expression of miR-21* or 203 may be mediated by the MAPK signaling pathway and AP-1, and both miR-21* and 203 have the effect on PTC aggressiveness. However, the absence of differential expressions of miR-21* and 203 between PTC tissues and paracancerous thyroid tissues on the miRNA microarray were probably due to the following: i) the small sample sizes of PTC tissues and paracancerous thyroid tissues for miRNA microarray analysis in the study; ii) the genotypic status (with/without BRAF or RAS gene mutations, RET/PTC, TRK, etc.) might be different among the 12 PTC tissues; and iii) these two miRNAs might be differentially expressed among different genotypic groups, while this hypothesis is needed to be further tested.
There are discordant findings in the literature in the relationship between BRAFV600E status and clinicopathological parameters: numerous studies (8, 11, 12, 13, 14) showed that BRAFV600E was significantly associated with poor clinicopathological parameters, while a small number of studies (5, 42) did not. In this study, we revealed significant relationships between poor clinicopathological features and the following: BRAFV600E positivity (using BRAFV600E-negative PTC tissues as the control group) and miR-21* over-expression (using paracancerous thyroid tissues as the control group). Interestingly, we also identified a significant association between BRAFV600E positivity and miR-21* over-expression. The molecular basis of this association should be explored in future studies.
In conclusion, our study has shed new light on the relationships between BRAFV600E status and miRNA expression, and between differentially expressed miRNA and clinicopathological feature in PTC. Although our findings need to be confirmed in studies of larger sample size, they may lay the foundation for the identification of a potential diagnostic biomarker or prognostic indicator of PTC.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by National Natural Science Foundation of China (no. 30872522); Natural Science Foundation of Guangdong Province (Key Program; no. 10251008901000004); and Foundation of Young Teachers training of Sun Yat-Sen University (no. 09ykpy46).
References
- 1↑
Xing M. BRAF mutation in thyroid cancer. Endocrine-Related Cancer 2005 12 245–262. (doi:10.1677/erc.1.0978).
- 2↑
Davies L, Welch HG. Increasing incidence of thyroid cancer in the United States, 1973–2002. Journal of the American Medical Association 2006 259 2164–2167. (doi:10.1001/jama.295.18.2164).
- 3↑
Mazzaferri EL, Jhiang SM. Long-term impact of initial surgical and medical therapy on papillary and follicular thyroid cancer. American Journal of Medicine 1994 97 418–428. (doi:10.1016/0002-9343(94)90321-2).
- 4↑
Johnson TL, Lloyd RV, Thompson NW, Beierwaltes WH, Sisson JC. Prognostic implications of the tall cell variant of papillary thyroid carcinoma. American Journal of Surgical Pathology 1988 12 22–27. (doi:10.1097/00000478-198801000-00003).
- 5↑
Liu RT, Chen YJ, Chou FF, Li CL, Wu WL, Tsai PC, Huang CC, Cheng JT. No correlation between BRAFV600E mutation and clinicopathological features of papillary thyroid carcinomas in Taiwan. Clinical Endocrinology 2005 63 461–466. (doi:10.1111/j.1365-2265.2005.02367.x).
- 6↑
Namba H, Nakashima M, Hayashi T, Hayashida N, Maeda S, Rogounovitch TI, Ohtsuru A, Saenko VA, Kanematsu T, Yamashita S. Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers. Journal of Clinical Endocrinology and Metabolism 2003 88 4393–4397. (doi:10.1210/jc.2003-030305).
- 7↑
Kim KH, Kang DW, Kim SH, Seong IO, Kang DY. Mutations of the BRAF gene in papillary thyroid carcinoma in a Korean population. Yonsei Medical Journal 2004 45 818–821.
- 8↑
Chakraborty A, Narkar A, Mukhopadhyaya R, Kane S, D'Cruz A, Rajan MG. BRAF V600E mutation in papillary thyroid carcinoma: significant association with node metastases and extra thyroidal invasion. Endocrine Pathology 2012 23 83–93. (doi:10.1007/s12022-011-9184-5).
- 9↑
Tufano RP, Teixeira GV, Bishop J, Carson KA, Xing M. BRAF mutation in papillary thyroid cancer and its value in tailoring initial treatment: a systematic review and meta-analysis. Medicine 2012 91 274–286. (doi:10.1097/MD.0b013e31826a9c71).
- 10↑
Nucera C, Porrello A, Antonello ZA, Mekel M, Nehs MA, Giordano TJ, Gerald D, Benjamin LE, Priolo C, Puxeddu E et al.. B-Raf(V600E) and thrombospondin-1 promote thyroid cancer progression. PNAS 2010 107 10649–10654. (doi:10.1073/pnas.1004934107).
- 11↑
Xing M. BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. Endocrine Reviews 2007 28 742–762. (doi:10.1210/er.2007-0007).
- 12↑
Elisei R, Ugolini C, Viola D, Lupi C, Biagini A, Giannini R, Romei C, Miccoli P, Pinchera A, Basolo F. BRAF(V600E) mutation and outcome of patients with papillary thyroid carcinoma: a 15-year median follow-up study. Journal of Clinical Endocrinology and Metabolism 2008 93 3943–3949. (doi:10.1210/jc.2008-0607).
- 13↑
Frasca F, Nucera C, Pellegriti G, Gangemi P, Attard M, Stella M, Loda M, Vella V, Giordano C, Trimarchi F et al.. BRAF(V600E) mutation and the biology of papillary thyroid cancer. Endocrine-Related Cancer 2008 15 191–205. (doi:10.1677/ERC-07-0212).
- 14↑
Kim SJ, Lee KE, Myong JP, Park JH, Jeon YK, Min HS, Park SY, Jung KC, Koo do H, Youn YK. BRAF V600E mutation is associated with tumor aggressiveness in papillary thyroid cancer. World Journal of Surgery 2012 36 310–317. (doi:10.1007/s00268-011-1383-1).
- 15↑
Bartel DP. MicroRNAs: genomics, biogenesis, mechanism and function. Cell 2004 116 281–297. (doi:10.1016/S0092-8674(04)00045-5).
- 16↑
Dalmay T. MicroRNAs and cancer. Journal of Internal Medicine 2008 263 366–375. (doi:10.1111/j.1365-2796.2008.01926.x).
- 17↑
Calin GA, Croce CM. MicroRNA signatures in human cancers. Nature Reviews. Cancer 2006 6 857–866. (doi:10.1038/nrc1997).
- 18↑
Nikiforova MN, Tseng GC, Steward D, Diorio D, Nikiforov YE. MicroRNA expression profiling of thyroid tumors: biological significance and diagnostic utility. Journal of Clinical Endocrinology and Metabolism 2008 93 1600–1608. (doi:10.1210/jc.2007-2696).
- 19↑
Sheu SY, Grabellus F, Schwertheim S, Worm K, Broecker-Preuss M, Schmid KW. Differential miRNA expression profiles in variants of papillary thyroid carcinoma and encapsulated follicular thyroid tumours. British Journal of Cancer 2010 102 376–382. (doi:10.1038/sj.bjc.6605493).
- 20↑
He H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S, Calin GA, Liu CG, Franssila K, Suster S et al.. The role of microRNA genes in papillary thyroid carcinoma. PNAS 2005 102 19075–19080. (doi:10.1073/pnas.0509603102).
- 21↑
Pallante P, Visone R, Ferracin M, Ferraro A, Berlingieri MT, Troncone G, Chiappetta G, Liu CG, Santoro M, Negrini M et al.. MicroRNA deregulation in human thyroid papillary carcinomas. Endocrine-Related Cancer 2006 13 497–508. (doi:10.1677/erc.1.01209).
- 22↑
Tetzlaff MT, Liu A, Xu X, Master SR, Baldwin DA, Tobias JW, Livolsi VA, Baloch ZW. Differential expression of miRNAs in papillary thyroid carcinoma compared to multinodular goiter using formalin fixed paraffin embedded tissues. Endocrine Pathology 2007 18 163–173. (doi:10.1007/s12022-007-0023-7).
- 23↑
Chen YT, Kitabayashi N, Zhou XK, Fahey TJ III, Scognamiglio T. MicroRNA analysis as a potential diagnostic tool for papillary thyroid carcinoma. Modern Pathology 2008 21 1139–1146. (doi:10.1038/modpathol.2008.105).
- 24↑
Lassalle S, Hofman V, Ilie M, Bonnetaud C, Puisségur MP, Brest P, Loubatier C, Guevara N, Bordone O, Cardinaud B et al.. Can the microRNA signature distinguish between thyroid tumors of uncertain malignant potential and other well-differentiated tumors of the thyroid gland? Endocrine-Related Cancer 2011 18 579–594. (doi:10.1530/ERC-10-0283).
- 25↑
Braun J, Hoang-Vu C, Dralle H, Hüttelmaier S. Downregulation of microRNAs directs the EMT and invasive potential of anaplastic thyroid carcinomas. Oncogene 2010 29 4237–4244. (doi:10.1038/onc.2010.169).
- 26↑
Kitano M, Rahbari R, Patterson EE, Xiong Y, Prasad NB, Wang Y, Zeiger MA, Kebebew E. Expression profiling of difficult-to-diagnose thyroid histologic subtypes shows distinct expression profiles and identify candidate diagnostic microRNAs. Annals of Surgical Oncology 2011 18 3443–3452. (doi:10.1245/s10434-011-1766-4).
- 27↑
Cahill S, Smyth P, Denning K, Flavin R, Li J, Potratz A, Guenther SM, Henfrey R, O'Leary JJ, Sheils O. Effect of BRAFV600E mutation on transcription and post-transcriptional regulation in a papillary thyroid carcinoma model. Molecular Cancer 2007 6 21–30. (doi:10.1186/1476-4598-6-21).
- 28↑
Li Volsi VA, Albores-Saavedra J, Asa SL, Baloch ZW, Sobrinho-Simoes M, Wenig B, DeLellis RA, Cady B. Papillary carcinoma. In World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Endocrine Organs, pp 57–66. Eds De Lellis RA, Lloyd RV, Heitz PU, Eng C. Lyon: IARC Press.2004
- 29↑
Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A eds. American Joint Committee on Cancer (2010). Thyroid. In AJCC Cancer Staging Manual, edn 7th, pp 87–96. Eds . New York: Springer.2010
- 30↑
Xu X, Quiros RM, Gattuso P, Ain KB, Prinz RA. High prevalence of BRAF gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines. Cancer Research 2003 63 4561–4567.
- 31↑
Kitano M, Rahbari R, Patterson EE, Steinberg SM, Prasad NB, Wang Y, Zeiger MA, Kebebew E. Evaluation of candidate diagnostic microRNAs in thyroid fine-needle aspiration biopsy samples. Thyroid 2012 22 285–291. (doi:10.1089/thy.2011.0313).
- 32↑
Shen R, Liyanarachchi S, Li W, Wakely PE Jr, Saji M, Huang J, Nagy R, Farrell T, Ringel MD, de la Chapelle A et al.. MicroRNA signature in thyroid fine needle aspiration cytology applied to “atypia of undetermined significance” cases. Thyroid 2012 22 9–16. (doi:10.1089/thy.2011.0081).
- 33↑
Keutgen XM, Filicori F, Crowley MJ, Wang Y, Scognamiglio T, Hoda R, Buitrago D, Cooper D, Zeiger MA, Zarnegar R et al.. A panel of four miRNAs accurately differentiates malignant from benign indeterminate thyroid lesions on fine needle aspiration. Clinical Cancer Research 2012 18 2032–2038. (doi:10.1158/1078-0432.CCR-11-2487).
- 34↑
Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W et al.. Mutations of the BRAF gene in human cancer. Nature 2002 417 949–954. (doi:10.1038/nature00766).
- 35↑
Kim SK, Kim DL, Han HS, Kim WS, Kim SJ, Moon WJ, Oh SY, Hwang TS. Pyrosequencing analysis for detection of a BRAFV600E mutation in an FNAB specimen of thyroid nodules. Diagnostic Molecular Pathology 2008 17 118–125. (doi:10.1097/PDM.0b013e31815d059d).
- 36↑
Nikiforova MN, Kimura ET, Gandhi M, Biddinger PW, Knauf JA, Basolo F, Zhu Z, Giannini R, Salvatore G, Fusco A et al.. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. Journal of Clinical Endocrinology and Metabolism 2003 88 5399–5404. (doi:10.1210/jc.2003-030838).
- 37↑
Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, Dhomen N, Hussain J, Reis-Filho JS, Springer CJ, Pritchard C et al.. Kinase-dead BRAF oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 2010 140 209–221. (doi:10.1016/j.cell.2009.12.040).
- 38↑
Sheu SY, Grabellus F, Schwertheim S, Handke S, Worm K, Schmid KW. Lack of correlation between BRAF V600E mutational status and the expression profile of a distinct set of miRNAs in papillary thyroid carcinoma. Hormone and Metabolic Research 2009 41 482–487. (doi:10.1055/s-0029-1215558).
- 39↑
Fujita S, Ito T, Mizutani T, Minoguchi S, Yamamichi N, Sakurai K, Iba H. miR-21 gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism. Journal of Molecular Biology 2008 378 492–504. (doi:10.1016/j.jmb.2008.03.015).
- 40↑
Sonkoly E, Wei T, Pavez Loriè E, Suzuki H, Kato M, Törmä H, Ståhle M, Pivarcsi A. Protein kinase C-dependent upregulation of miR-203 induces the differentiation of human keratinocytes. Journal of Investigative Dermatology 2010 130 124–134. (doi:10.1038/jid.2009.294).
- 41↑
Domenzain-Reyna C, Hernández D, Miquel-Serra L, Docampo MJ, Badenas C, Fabra A, Bassols A. Structure and regulation of the versican promoter: the versican promoter is regulated by AP-1 and TCF transcription factors in invasive human melanoma cells. Journal of Biological Chemistry 2009 284 12306–12317. (doi:10.1074/jbc.M807108200).
- 42↑
Kim TY, Kim WB, Song JY, Rhee YS, Gong G, Cho YM, Kim SY, Kim SC, Hong SJ, Shong YK. The BRAF mutation is not associated with poor prognostic factors in Korean patients with conventional papillary thyroid microcarcinoma. Clinical Endocrinology 2005 63 588–593. (doi:10.1111/j.1365-2265.2005.02389.x).
