miR-146b is Highly Expressed in Adult Papillary Thyroid Carcinomas with High Risk Features Including Extrathyroidal Invasion and the BRAF V600E Mutation

Abstract

Background:

Papillary thyroid carcinoma (PTC) are clinicopathogenetically heterogeneous. Micro-RNAs (miRNAs) are involved in the pathogenesis of diverse human cancers, including PTC. Information regarding associations between clinicopathological features of PTC with the expression of specific miRNAs, however, is sparse. In this study, we compared expression of deregulated miRNAs in PTCs to assess this was associated with selected clinicopathogenetic features.

Methods:

We analyzed the expression levels of three reported deregulated miRNAs (miR-221, miR-222, and miR-146b) using quantitative real-time polymerase chain reaction in 100 cases of PTCs with distinct clinicopathogenetic characteristics and 16 paired normal controls. The tumor samples were categorized into low- and high-risk groups on the basis of the tumor-node-metastasis staging system.

Results:

The expression levels of miR-221, miR-222, and miR-146b were significantly associated with extrathyroidal invasion (p = 0.013, 0.05, and 0.003, respectively). The expression levels of miR-221 and miR-146b were significantly higher in the high-risk PTC group (p = 0.01 and 0.042, respectively). The miR-146b expression levels in PTCs with BRAF mutation were significantly higher than those without this mutation (p < 0.0001). There were no other associations between the expression of these miRNAs and other clinicopathological parameters.

Conclusions:

Our results show the potential importance of miR-221, miR-222, and miR-146b in determining the aggressive properties of PTCs and highlight the need to identify the gene targets of these miRNAs.

Introduction

P apillary thyroid carcinoma (PTC) is clinicopathogenetically heterogeneous. The biological features and clinical outcomes of this disease vary considerably. Certain clinical and pathological features such as advanced age at diagnosis, larger primary tumors (≥3 cm), extrathyroidal invasion, distant metastases, and aggressive histological variants (e.g., the tall-cell variant of papillary cancer) have been associated with a worse prognosis of this disease (1 –3). Genetic mutations in four well-known proto-oncogenes (RET rearrangement, NTRK1 rearrangement, RAS point mutation, and BRAF point mutation) act through the RET/PTC(TRK)–RAS–BRAF–MEK–MAPK signaling pathway that contributes to the pathogenesis of PTCs (4). Many, but not all, published reports indicate that PTCs harboring mutations in BRAF, which is the most common genetic alteration in PTCs (50% on average), exhibit more aggressive properties such as cervical lymph node (LN) metastases, extrathyroidal invasion, advanced stage at diagnosis, and tumor recurrence (5).

Micro-RNAs (miRNAs) are small noncoding RNA molecules that function as negative regulators of gene expression by binding to the 3′-untranslated region of target mRNAs, and by blocking the translation or degradation of the mRNAs (6 –8) that mediate various pathophysiological processes. The deregulation or aberrant expression of miRNAs has recently been implicated in the pathogenesis of diverse human cancers, including differentiated and undifferentiated thyroid cancers (9 –14). Although the exact function of many miRNAs remains to be elucidated, identification of the most significant and informative aberrantly expressed miRNAs in cancer tissues would lead to a better understanding of gene regulation in cancers (15). Recent studies on miRNA deregulation have demonstrated an increased aberrant miRNA expression (particularly, miR-222, miR-221, and miR-146b) in PTCs compared with normal thyroid tissues (9,10,13,14). These data indicated the miRNA signature associated with PTCs and suggested that miRNA deregulation is an important event in thyroid cell transformation. However, the target genes regulated by these important miRNAs remain largely unknown and little has been reported on the association of the clinicopathogenetic features of PTC with specific miRNA expression.

In this study, we sought to further elucidate the differences in the expression patterns of several miRNAs in patients with distinct clinicopathogenetic features of PTCs, so as to elucidate whether deregulated expression of certain specific miRNAs has any association with the clinicopathogenetic features of this disease.

Materials and Methods

Tumor samples and patient information

Details of the clinicopathogenetic features of the PTCs in this study are presented in Table 1. Age range of patients was 18–85 years, and the male:female ratio was 3:7. We analyzed fresh thyroid samples from 100 adult patients with PTC and 16 paired unaffected thyroid tissues. These samples were selected based on their clinicopathogenetic features and tissue availability from our prospective tissue collections by one of the authors (F.-F.C.) since 1997. The samples were snap-frozen in liquid nitrogen at the time of thyroidectomy and subsequently stored at −80°C. The details of the clinical data collection, histological examination, tumor subtyping, tumor-node-metastasis classification, and oncogene status determination (RET rearrangement, NTRK1 rearrangement, and BRAF point mutation) for these samples have been described previously (16). Of the 100 PTC cases, 63 have been reported elsewhere (16). Patients who were less than 45 years old and had stage I PTC, or 45 years or more with stage I or II PTC were defined as low-risk group according to the American Joint Commission on Cancer–International Union Against Cancer criteria. The remaining patients were defined as high-risk group (17).

Table 1.

Clinicopathogenetical Features of Papillary Thyroid Carcinomas in This Study (n = 100)

Clinicopathogenetic features	Number
Age at the time of diagnosis (years)	44.7 ± 15.7
Sex (male:female)	30:70
Tumor size (cm)	2.8 ± 1.3
Oncogene status
BRAF	46
TRK	1
RET/PTC	12
Distant metastasis	4
Tumor location
Unilateral	69
Bilateral	31
TNM staging (AJCC)
I	58
II	4
III	26
IV	12
Multicentricity	53
Calcification	59
Psammoma body	46
Cervical LN metastasis	37
Tumor subtype
Classic	59
Follicular	14
Tall cell	18
Encapsulated	6
Oncocytic	3
Solid trabecular	1
Diffuse sclerosing	1
Extrathyroidal invasion	46

PTC, papillary thyroid carcinoma; TNM, tumor-node-metastasis; AJCC, American Joint Commission on Cancer; LN, lymph node.

This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. Informed consent was obtained from the patients included in this study.

RNA extraction

Total RNA was extracted from surgical specimens using Trizol reagent (Invitrogen). In brief, 0.5 mL Trizol reagent was added to 20 mg of each surgical specimen to extract RNA without any DNA and protein contamination. The samples were thoroughly vortexed and 0.1 mL chloroform (Scharlau, Barcelona, European Union) was added for phase separation. The samples were then centrifuged at 12,000 g for 10 minutes and the upper aqueous phase was transferred to fresh diethyl phosphorocyanidate-treated Eppendorf tubes to which an equal volume of isopropanol (Merck KGaA) was added for RNA precipitation at −20°C for 1 hour. RNA was harvested by centrifugation at 12,000 g for 10 minutes at 4°C, followed by 75% ethanol precipitation (Merck KGaA).

Quantitative reverse transcription–polymerase chain reaction

The single-tube TaqMan miRNA assay (Applied Biosystems) was used to quantify the mature miRNA expression levels on an Applied Biosystems 7500 system according to the manufacturer's instructions. The expression levels of individual miRNAs—miR-221 (part number, 4427975; ID, 000524), miR-222 (part number, 4427975; ID, 000525), and miR-146b (part number, 4427975; ID, 001097)—were analyzed using miRNA sequence-specific primers (Applied Biosystems). cDNA templates were obtained by reverse transcribing 50 ng of total RNA using the TaqMan MicroRNA Reverse transcription Kit (Applied Biosystems). The reaction conditions for the procedure were as follows: one cycle of 30 minutes at 16°C, 30 minutes at 42°C, and 5 minutes at 85°C. The quantitative reverse transcription–polymerase chain reaction (RT-PCR) was performed with 2 μL of 10-fold-diluted cDNA using 10 μL of TaqMan Universal PCR Master Mix and 1 μL of TaqMan miRNA sequence-specific probe (Applied Biosystems) or U6 probe (Applied Biosystems). The PCR conditions used were as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 1 minute. The integrity of the miRNA and the efficiency of the RT-PCR in each sample were confirmed by performing PCR for the endogenous control U6 small RNA. Using the same RNA quantity for the control PCR, the threshold cycle (Ct) range for the control U6 small RNA ranged from 28 to 32 cycles. All RT-PCR for quantifying the expression levels of miR-221, miR-222, and miR-146b were performed twice in different run for each sample, and the relative quantities for each sample were determined by the comparative Ct method (2^−ΔΔCt) (18), where ΔCt = (Ct_miRNA − Ct_U6). Mean Ct values for each sample were calculated and then normalized against the corresponding control U6 Ct values. The Ct values for miR-221, miR-222, and miR-146b cluster were within two amplification cycles for all samples. The data were presented in fold increase in miRNA expression in PTC samples compared with the expression levels in normal thyroid tissue samples.

Statistical analysis

Data have been indicated as medians (range). All statistical analyses were performed using the Statistical Package for Social Science program (SPSS for Windows, version 13.0). The difference in miRNA expression levels between PTC samples and matched normal tissue samples or subgroups classified according to the different clinical and pathological features was analyzed using the Wilcoxon signed-rank test and Mann–Whitney U-test. Differences of p < 0.05 were considered statistically significant.

Results

Of the 100 cases of PTC, 46 had heterozygous T1799A BRAF mutation (BRAF^V600E) and 12 had RET rearrangements. Of the RET rearrangements, 6 involved RET/PTC1, 1 involved RET/PTC2, 4 involved RET/PTC3, and 1 involved ELKS-RET rearrangement. There was a single instance of TRK-T2 rearrangement out of the 100 PTC cases.

To determine whether any of the clinicopathogenetic features of the PTCs were associated with the differential expression levels of miRNAs, we selected clinical samples (one low risk and one high risk) for miRNA microarray expression analysis (Ambion) to evaluate miRNA profile differences. The top three miRNAs (miR-221, miR-146b, and miR-222) showed dramatic overexpression (49.03 ± 2.54X in miR-221, 22.97 ± 0.92X in miR-146b, and 21.25 ± 12.87X in miR-222) in high-risk PTC tumor compared with the low-risk PTC sample. Further, the expression patterns of three miRNAs (miR-221, miR-222, and miR-146b) were evaluated by quantitative RT-PCR. These deregulated miRNAs have been reported to be upregulated in PTC as compared with normal thyroid tissue (9,10,13,14). Analysis of these three miRNA (miR-221, miR-222, and miR-146b) expression levels in the 100 PTCs, which were selected on the basis of different clinicopathogenetic features, and 16 matched normal tissues revealed that there was significant overexpression of miR-221, miR-222, and miR-146b in PTCs compared with normal tissue (p = 0.01, 0.049, and 0.001, respectively) (Fig. 1). The expression levels of miR-221, miR-222, and miR-146b were significantly associated with extrathyroidal invasion (p = 0.013, 0.05, and 0.003, respectively) (Table 2). The expression levels of miR-221 and miR-146b were significantly higher in the high-risk PTC group than in the low-risk group as revealed by the tumor-node-metastasis staging system (p = 0.01 and 0.042, respectively). The miR-146b expression levels in PTCs with BRAF mutation were significantly higher than those without this mutation (p < 0.0001). miR-146b expression levels in female patients were also lower than those in male patients (p = 0.038). Among the four groups stratified on the basis of risk group and BRAF mutation status, the high-risk group patients with concomitant BRAF mutation showed the highest increase in miR-146b expression compared with the other groups (Fig. 2).

FIG. 1.

Quantitative reverse transcription–polymerase chain reaction analysis of miR-221, miR-222, and miR-146b in PTCs (n = 16) and paired normal tissue. The fold change values indicate the relative change in the expression levels between samples and its internal control (U6), assuming that the value of U6 expression level of each sample was equal to 1. PTC, papillary thyroid carcinoma.

FIG. 2.

miR-146b expression levels in PTCs stratified on the basis of risk group and BRAF mutation status. Fold change in miRNA expression was calculated relative to normal thyroid tissue. LW, low-risk group with wild-type BRAF; HW, high-risk group with wild-type BRAF; LM, low-risk group with BRAF mutation; HM, high-risk group with BRAF mutation; LW versus HW, HW versus LM, and LM versus HM: for all, p > 0.05.

Table 2.

Association of Micro-RNAs (miR-221, miR-222, and miR-146b) with Different Clinicopathogenetical Features in Papillary Thyroid Carcinomas

miRNAs			221		222		146
Clinicopathogenetical features		n	–(ΔCt) ^a	p	–(ΔCt)	p	–(ΔCt)	p
Age at the time of diagnosis (years)	<45	54	7.5 (2.3–13.4)	0.278	9.1 (3.7–16.4)	0.691	9.3 (3.2–17.7)	0.233
	≥45	46	8.1 (2.3–13.7)		9.2 (3.7–16.4)		10.2 (3.2–17.7)
Sex	Male	30	7.7 (2.3–11.1)	0.513	9.3 (3.7–13.0)	0.248	10.1 (3.2–14.5)	0.038
	Female	70	7.9 (2.3–13.7)		8.7 (4.3–16.4)		8.5 (2.7–17.7)
Tumor size (cm)	<3	57	7.4 (2.3–13.4)	0.155	8.9 (3.7–13.0)	0.085	9.5 (4.4–13.8)	0.783
	≥3	40	8.6 (2.3–13.7)		9.4 (4.3–16.4)		10.0 (2.7–17.7)
Tumor staging (AJCC)^b	Low risk	62	7.3 (2.3–13.4)	0.013	8.9 (4.3–13.6)	0.076	9.1 (2.7–13.8)	0.042
	High risk	38	8.6 (2.3–13.7)		9.5 (4.9–16.4)		10.3 (3.2–17.7)
BRAF mutation	No	54	7.3 (2.3–13.0)	0.062	9.0 (3.7–13.6)	0.085	8.6 (2.7–12.3)	0.000
	Yes	46	8.6 (2.9–13.7)		9.3 (5.3–16.4)		11.2 (3.2–17.7)
Cervical LN metastasis	No	63	7.5 (2.3–13.4)	0.133	8.9 (3.7–13.6)	0.058	10.1 (3.6–14.8)	0.176
	Yes	37	8.4 (2.9–13.7)		9.4 (25.7–16.4)		10.2 (2.7–17.7)
Multicentricity	No	47	7.5 (2.3–13.7)	0.501	8.9 (3.7–16.4)	0.641	10.5 (3.2–17.7)	0.602
	Yes	53	7.8 (2.3–13.0)		9.3 (4.3–13.6)		9.7 (2.7–14.5)
Psammoma body	No	54	7.7 (2.3–13.4)	0.966	10.1 (3.7–13.6)	0.943	10.1 (3.2–17.7)	0.800
	Yes	46	7.9 (2.3–13.7)		9.3 (4.3–16.4)		10.5 (2.7–14.5)
Extrathyroidal invasion	No	54	7.1 (2.3–13.4)	0.013	8.8 (3.7–13.6)	0.050	8.8 (2.7–14.8)	0.003
	Yes	46	8.6 (2.9–13.7)		9.5 (5.7–16.4)		10.4 (3.2–17.7)

The data are shown as median (range).

−ΔCt = −(Ct_miRNA − Ct_U6).

The low-risk group was defined as those patients who were less than 45 years old and had stage I PTC and those patients who aged 45 years or more with stage I or II PTC according to the AJCC. The remaining patients were defined as high-risk group.

miRNA, micro RNA; Ct, threshold cycle.

There were no significant difference in the expression levels of any of these miRNAs based on other clinicopathologic parameters, including age at the time of diagnosis, tumor size, cervical LN metastases, multicentricity, calcification, and the presence of psammoma body (Table 2). Since PTCs harboring mutations in BRAF have more aggressive properties (5), we correlated the BRAF mutation status with clinicopathologic features of PTCs in this study. No significant correlation was observed between BRAF mutation and sex, age at the time of diagnosis, tumor size, multicentricity, cervical LN metastases, extrathyroidal invasion, clinical stage of cancer, or distant metastases (data not shown). These findings confirmed our previous results (16).

Discussion

In this study, we have reported the association between the expression levels of miR-221, miR-222, and miR-146b and various clinicopathogenetic features of PTCs. We showed that the expression levels of miR-221, miR-222, and miR-146b were significantly higher in PTCs with extrathyroidal invasion than in those without the invasion. The expression levels of miR-221 and miR-146b were also significantly higher in the high-risk group than in the low-risk group. However, only miR-146b had significantly higher expression in PTCs harboring BRAF mutation than those without the mutation. To our knowledge, this is the first report that demonstrates the association between miRNA expression and clinicopathogenetic features of PTCs in humans. Our findings are consistent with those of certain recent reports that correlate the levels of miRNA expression with the clinicopathological parameters in breast cancers (19), lung cancers (20), and pituitary adenomas (21).

Overexpression of miR-221, miR-222, and miR-146b in PTCs compared to the matched normal thyroid tissue has been demonstrated in several previous studies (9,10,13,14) and also in the reported cases of our lab. In this study, we have shown that these miRNAs are not only associated with PTC carcinogenesis, which was consistent with other reports (9,10,14), but also related to certain clinicopathogenetic features of PTCs. Our findings are analogous to those reported by Ma and Weinberg, in which miR-10b (1 of the 29 miRNAs that are known to have altered expression levels in primary breast carcinomas compared with normal mammary tissues) specifically promotes tumor cell invasion (22). It would be possible to speculate that altering miRNA expression levels might be an efficient strategy for cancer cells to simultaneously alter the expression profiles of a series of genes. Our finding that specific miRNAs are associated with certain clinicopathogenetic characteristics supports the hypothesis that alterations in miRNA expression may provide cancer cells with a selective growth advantage, allowing them to develop aggressive properties.

Cancer cells often express a constitutively active nuclear factor-kappaB (NF-κB) to promote survival, proliferation, and metastatic potential by mechanisms that remain largely unknown (23,24). It has been shown that BRAF^V600E activates not only MAPK but also the NF-κB signaling pathway in human thyroid cancer cells, which confers apoptotic resistance and promotes cancer invasion, consistent with the poor prognosis of human PTCs with BRAF mutation. It was demonstrated that both miR-146a and miR-146b are known targets of NF-κB and are up-regulated following toll-like receptor (TLR)2, TLR4, or TLR5 ligation as well as in response to tumor necrosis factor (TNF)α or interleukin (IL)-1β stimulation in stimulated monocytes (25). Similarly, a recent study showed that miR-146 is expressed in rheumatoid arthritis synovial tissue, and its expression is induced by TNFα and IL-1β stimulation (26). The significant association between higher expression level of miR-146b and PTCs harboring BRAF mutation or with extrathyroidal invasion or with the high-risk group patients is compatible with the hypothesis that miR-146b might be a downstream gene of BRAF mutation and might influence the predisposition to extrathyroidal invasion and lead to an advanced PTC stage. However, a few notes of caution are appropriate. First, it has yet to be confirmed whether PTC cells carrying BRAF mutation are able to upregulate miR-146b by activating NF-κB. Further studies are required to address this issue. Second, Taganov et al. (25) identified IL-1-receptor-associated kinase and TNF-receptor-associated factor 6 to be the direct targets of miR-146a and miR-146b expression as a part of an NF-κB-induced negative feedback loop, indicating that miR-146a/b may be important negative regulators of the TLR signaling pathway (25,27,28). On the basis of this observation, Bhaumik et al. (29) demonstrated that the expression of miR-146a/b in a highly metastatic human breast cancer cell line suppresses NF-κB activity and reduces its metastatic potential. This observation was in direct contrast to our finding of higher miRNA-146b expression associated with extrathyroidal invasion and advanced tumor stage. The reasons for the apparent difference are not known. However, this finding may not be surprising in that some miRNAs behave in cancer cells in a dual mode, behaving as oncogenes or tumor suppressors depending on tissue type and specific targets (30,31). Consistent with this, miR-146a was found to promote cell proliferation in human cervical cancer cell lines (30,31).

Correlation of BRAF mutation with PTC clinicopathologic features yields controversial results. Our previous study demonstrated no correlation between BRAF mutation and the clinicopathologic features of patients with PTCs in Taiwan (16). In this study, we confirm our previous findings. Our observations that higher expression levels of miR-221 and miR-146b were significantly associated with extrathyroidal invasion and advanced tumor stage but only miR-146b was significantly associated with BRAF mutation may partially account for this controversy in results.

Conclusions

Our results highlight the importance of miR-221, miR-222, and miR-146b in determining the aggressive properties of PTCs. Although further studies are required to examine these correlations, our data provide insight into the role of these miRNAs and may contribute to the identification of the potential gene targets of these miRNAs.

Footnotes

Acknowledgments

The authors would like to thank the National Science Council of the Republic of China, Taiwan (Contract no. NSC95-2314-B-182A-184 and NSC96-2314-B-182A-036-MY2), and Chang-Gung Memorial Hospital (CMRPG83038) for financially supporting this research.

Disclosure Statement

The authors declare that no competing financial interests exist.

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