J C E M
O N L I N E
Hot Topics in Translational Endocrinology—Endocrine Care
Prognostic Implications of miR-146b Expression and
Its Functional Role in Papillary Thyroid Carcinoma
Chen-Kai Chou, Kuender D. Yang, Fong-Fu Chou, Chao-Cheng Huang,
Yueh-Wen Lan, Ya-Fang Lee, Hong-Yo Kang,* and Rue-Tsuan Liu*
Division of Metabolism (C.-K.C., Y.-W.L., Y.-F.L., R.-T.L.), Department of Internal Medicine; Departments
of Surgery (F.-F.C.) and Pathology (C.-C.H.), Chang Gung Memorial Hospital-Kaohsiung Medical Center,
Kaohsiung Hsien, Taiwan; and Graduate Institute of Clinical Medical Sciences (C.-K.C., H.-Y.K.), Chang
Gung University, Taiwan; and Department of Medical Research (K.D.Y.), Show Chwan Memorial
Hospital in Chang Bing, Changhua, Taiwan
Context: Recent studies suggest that miR-146b deregulation in papillary thyroid carcinoma (PTC)
was associated with advanced tumor characteristics. However, the influence of miR-146b expression on the prognosis of PTC remains unknown. We sought to correlate tumor expression levels of
miR-146b with the prognosis of a previously reported PTC cohort and reveal the underlying mechanisms via a PTC cell line model.
Methodology: Expression levels of miR-146b were assessed via quantitative real-time PCR in 71
cases of PTC with distinct clinico-pathogenetic characteristics. All patients were classified into the
disease-free or active disease group, based on their medical records at the end of the follow-up
period. In vitro gain-of-function experiments were performed in a BCPAP human papillary thyroid
cancer cell line model, which harbored the homozygous mutation of BRAF. BCPAP cells were
transfected with a mimic-miR-146b and nonspecific microRNA (miRNA) control to determine
whether miR-146b overexpression promotes cell migration and invasion. Proliferation assay, colony formation assay, and chemotherapy-induced apoptosis were also determined.
Results: Multivariate logistic regression analysis demonstrated advanced tumor stage, presence of
cervical lymph node metastasis, and miR-146b expression were independent risk factors for poor
prognosis in PTC. Patients with higher miR-146b expression levels had significantly poorer overall
survival compared with those with lower miR-146b levels. The associated hazard ratio was 3.92
(95% confidence interval, 1.73– 8.86, log-rank P ⬍ .05). Overexpression of miR-146b significantly
increased cell migration and invasiveness. Furthermore, miR-146b also increased resistance to
chemotherapy-induced apoptosis.
Conclusions: Our results suggest that miR-146b is a novel prognostic factor of PTC. Furthermore,
in vitro functional studies provided the mechanistic explanation for miR-146b in tumor aggressiveness. These results enhance understanding of the molecular mechanisms involved in tumor
aggressiveness in PTC, provide new prognostic biomarkers, and ultimately offer new leads for
developing therapies for PTC. (J Clin Endocrinol Metab 98: E196 –E205, 2013)
P
apillary thyroid carcinoma (PTC) is a clinico-pathogenetically heterogeneous disease (1–3). Although
most PTCs could be managed successfully with a combination of radioiodine and levothyroxine treatment after
complete thyroidectomy, tumors with more aggressive
phenotype are associated with morbidity and mortality
(4). Clearly, understanding the molecular events involved
in the initiation and progression of papillary thyroid can-
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
doi: 10.1210/jc.2012-2666 Received July 2, 2012. Accepted November 29, 2012.
First Published Online December 21, 2012
* R.-T.L. and H.-Y.K. contributed equally to this study.
Abbreviations: CI, Confidence interval; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; FNA, fine-needle aspiration; HR, hazard ratio; LN, lymph node; miRNA, microRNA;
PTC, papillary thyroid carcinoma; Tg, thyroglobulin; XTT, 2,3-bis-(2-methoxy-4nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.
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cer will be helpful for the identification of novel diagnostic, prognostic, and therapeutic targets.
It is evident that, in addition to alterations in proteinencoding genes, abnormalities in non-protein-coding
genes can also contribute to cancer pathogenesis (5). MicroRNAs (miRNAs), a class of non-protein-coding RNAs,
are believed to function as negative regulators of gene expression by binding to the 3⬘-untranslated region of target
mRNAs and causing a block of translation or degradation
of mRNAs. Indeed, miRNAs have also been implicated in
the regulation of a variety of cellular processes, including
apoptosis (6), hematopoietic differentiation (7), and metastasis (8). These miRNAs may function as tumor suppressors or oncogenes in various cancers (8 –10). Although the exact mechanisms of action of various
miRNAs are yet to be elucidated, the identification of the
most significant and informative aberrantly expressed
miRNAs would lead to a better understanding of gene
regulation in tumorigenesis and tumor behavior.
Recent studies on miRNA deregulation have demonstrated that there is an increase in aberrant miRNA expression, particularly miR-222, miR-221, and miR-146b,
in PTC vs normal thyroid tissue (11–13). These data indicate that miRNA signature is associated with PTC and
that miRNA deregulation is an important event in thyroid
cell transformation. In our previous study (14), overexpression of miR-146b is not only associated with PTC
carcinogenesis but also related to extrathyroidal invasion,
advanced stages of PTC, and BRAF mutation in PTC.
These results highlight the importance of miR-146b in
determining the aggressiveness of PTC and may contribute
to the identification of the potential gene targets related to
tumor aggressiveness in PTC. Our findings were subsequently confirmed by Yip et al (15). Recent studies also
have reported the altered expression of miR-146b in several kinds of solid tumors other than PTC. In patients with
triple-negative sporadic breast cancers, down-regulation
of BRCA1 expression via miR-146b was confirmed,
which led to an increase in cellular proliferation of mammary cell lines (16). In another tissue microarray experiment, miR-146b was shown to be a poor prognostic
marker in patients with oral squamous cell carcinoma
(17). In addition, Raponi et al (18) demonstrated that
miR-146b was the most robust factor in predicting the
prognosis of patients with lung squamous-cell carcinoma.
However, it has not yet been confirmed whether the deregulation of this miRNA in PTC exerts a causative effect
in regard to tumor aggressiveness.
The aim of the present study was to determine the influence of miR-146b on tumor aggressiveness and patient
prognosis. Herein, we evaluated the prognostic efficacy of
miR-146b in a previous published PTC cohort (14) and
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determined the mechanisms by which miR-146b influences PTC aggressiveness via a BRAF mutation-harboring
PTC cell line.
Materials and Methods
Tumor samples and patient information
The present study assessed tissue samples previously obtained
from the thyroidectomies of patients between 1997 and 2005.
We retrospectively reviewed the clinical records and histological
examinations of these patients, who received standard treatment
for PTC, including surgery, radioactive iodine, and thyroid hormone therapy, and were followed up over a duration of 127.0 ⫾
29.8 months at the Chang Gung Memorial Hospital-Kaohsiung
Medical Center.
Of 100 patients whose tumor samples were obtained as previously reported (14), 25 cases who did not receive postoperative
radioactive iodine therapy and the 4 cases who did not receive
subsequent whole-body 131I scanning and short period of follow-up (less than 5 years) were excluded due to failure of recurrence/persistence status determination. Seventy-one patients
who had sufficient follow-up clinical information were used for
prognostic analyses. Tissue samples were snap-frozen in liquid
nitrogen at the time of total thyroidectomy and subsequently
stored at ⫺80°C. Thyroid cancer recurrence and/or persistence
was defined as a positive radioiodine whole-body scan after curing the initial disease and/or an investigation of a tumor mass,
which cytologically or pathologically confirmed the thyroid cancer. The clinical follow-up interval for patients that had a recurrence and/or persistence of cancer was defined as the time from
the thyroidectomy to the detection of their tumor recurrence
and/or persistence. All patients were classified as disease-free or
active disease based upon their medical records at the end of the
follow-up.
In the disease-free patients, the clinical follow-up interval was
defined as the time from the thyroidectomy until May 2011. The
BRAF mutation was detected, as previously reported (19). Details concerning clinical data collection, tumor node-metastasis
classification, and BRAF status for these samples have been previously described elsewhere (14). Patients that were ⬍45 years
old and had stage I PTC or those that were ⱖ45 years old with
stage I or II PTC were defined as a low-risk group according to
the American Joint Commission on Cancer-International Union
Against Cancer criteria. The remaining patients were defined as
a high-risk group (20). This study was approved by the Institutional Review Board of the Chang Gung Memorial Hospital.
Informed consent was obtained from all patients included in this
study.
Thyroglobulin (Tg) assay
Serum Tg levels were measured by immunoradiometric assay
(Tg IRMA, CIS-Bio International, Gif-sur-Yvette, France). The
interassay coefficients of variation were 14.6%, 4.6%, and 6.2%
at low, median, and high concentrations, respectively. The intraassay coefficients of variation were 7%, 2.4%, and 1.8% at
low, median, and high concentrations, respectively. The detection limit was 0.2 ng/mL. TSH-stimulated Tg was checked under
withdrawal of thyroid hormone completely for 4 weeks.
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MiR-146b and Tumor Aggressiveness in PTC
Cell culture
The human thyroid cancer cell line BCPAP, which harbors the
homozygous mutation of BRAF, was kindly provided by Professor Dumont, Universite Libre de Bruxelles, Belgium. The cell
line was routinely cultured in RPMI 1640 containing 10% fetal
bovine serum (FBS), 100 U/mL penicillin, 100 g/mL streptomycin, and 2 mM L-glutamine (GIBCO, Rockville, MD) at 37°C in
a humidified chamber containing 5% CO2.
RNA extraction and quantitative RT-PCR
Total RNA was extracted from surgical specimens or the
BCPAP cell line using Trizol reagent (Invitrogen, Carlsbad, CA).
For miRNA expression analysis, RNA was reverse transcribed
using a TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA) and RT primers were provided with the
miR-146b-5p TaqMan miRNA assay (PN4373178; Applied
Biosystems) according to the manufacturer’s instructions. Experimental details were described in a previous study (14).
miRIDIAN miRNA mimic and delivery
The cells were seeded (3 ⫻ 105 cells per well in 6-well plate)
in antibiotic-free medium for 24 hours prior to transfection.
DharmaFECT transfection reagent (Dharmacon RNA Technologies, Lafayette, CO) was used to transfect cells with mimicmiR-146b (Dharmacon RNA Technologies) for 24 hours at 50%
confluence following the manufacturer’s instructions. The
miRIDIAN miRNA mimic negative control (Dharmacon RNA
Technologies) was used as control. Samples were collected after
24 hours of miRNA mimic transfections for quantification of
miRNA.
Colony formation assay
BCPAP cells were transfected with the mimic-miR-146b or
the negative control for 24 hours, and then 1000, 500, and 100
cells were seeded separately in 6-well plates for colony formation. After 14 days, colonies were fixed and stained with a mixture of 6% glutaraldehyde and 0.5% crystal violet. Only if a
single colony contained more than 50 cells was it scored using a
microscope. Each assay was performed in duplicate on two independent occasions.
Cell-proliferation assay
For determination of cell proliferation/viability, the numbers
of viable cells were estimated based on the 2,3-bis-(2-methoxy4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT)
cell proliferation kit (Roche Diagnostics, Mannheim, Germany).
BCPAP cells were transfected with the mimic-miR-146b or the
negative control for 24 hours, and then 5000 cells per well were
seeded in 96-well plates. Fifty microliters of XTT mixture reagent was added to each well, and the absorbance was measured
at different time points by the absorbance at 492 nm with an
ELISA reader (Victor2 1420 multilabel counter; PerkinElmer
Life Sciences, Waltham, MA).
Chemotherapy-induced apoptosis: assessment of
apoptosis by annexin-V and propidium iodide
staining
Cell lines were transfected with mimic-miR-146b or nonspecific control at 50% confluence. After 24 hours, the cells were
treated with 0.015 M cyclophosphamide for 24 hours. The cells
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were washed with PBS once and resuspended in 98 ␮L of binding
buffer (annexin-V/fluorescein isothiocyanate [FITC] apoptosis
detection kit; Strong Biotech Corp., Taipei, Taiwan). Then, 2 ␮L
of annexin-V/FITC and 2 ␮L of propidium iodide were added to
the binding buffer, and the tubes were incubated at room temperature for 15 min in the dark. Thus, the fluorescence of the cells
was determined immediately with a flow cytometer (BD LSR II
cytometer; BD Biosciences, Bedford, MA). Apoptotic cells could
be stained with either the propidium iodide solution or
annexin-V/FITC.
In vitro migration and invasion assay
We used a Transwell insert (24-well insert, pore size 8 ␮m;
Corning, Corning, NY) to determine the effect of miR-146b on
PTC cell migration in vitro. For Transwell migration assays, 5 ⫻
104 cells were plated in the top chamber with the noncoated
membrane. The transfected BCPAP cells were first starved in
medium without FBS overnight, and the cells were resuspended
in the FBS-free medium and placed in the top chambers in duplicate. The lower chamber was filled with 10% FBS as the chemoattractant and incubated for 24 hours for the migration assay.
For the invasion assay, the inserts were previously coated with
extracellular matrix gel from Engelbreth-Holm-Swarm mouse
sarcoma (BD Biosciences). At the end of the experiments, the cells
on the upper surface of the membrane were removed using cotton
buds, and the cells on the lower surface of the insert were fixed
and stained with 10% Giemsa stain. Five visual fields of each
insert were randomly chosen and photographed under a light
microscope at ⫻200 magnification. The cells in the photographs
were counted, and the data were summarized as means ⫾ SDs
and presented as a percentage of controls.
Statistical analysis
Data are presented as either a percentage or mean ⫾ SD. All
statistical analyses were performed using the Statistical Package
for Social Science program (SPSS for Windows, version 17.0).
Clinical features and miR-146b expression differences in active
disease and disease-free subgroups were determined via the Pearson’s ␹2 test for categorical variables or the Student’s t test for
continuous variables. The association between PTC’s prognosis
and clinical parameters was first analyzed by univariate analysis,
and those significantly differing were enrolled in multiple logistic
regression with a forward stepwise procedure to identify independent risk factors for prognosis in PTCs.
Kaplan-Meier curves were constructed to determine the patient disease-free survival rates. The Cox score was used to determine each patient’s risk of disease-free survival. Kaplan-Meier
survival plots and log-rank tests were used to assess the differences in disease-free survival of the different subgroups of PTC
patients. Kaplan-Meier plots were generated via the Statistical
Package for Social Science program. P ⬍ .05 was considered
statistically significant.
Results
Expression of miR-146b presents as a novel
prognostic marker in PTC’s outcome
Among the 71 eligible cases, 30 were classified into the
active disease (ie, 2 died and 28 presented with persistent
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TABLE 1. Clinical and Histological Features of Patients With PTC by Groupa
Subgroup by Prognosis
Clinical Feature
Age at diagnosis, y
Sex, % male
miR-146bc
Tumor size, cm
Tumor stage, % high-riskd
Cervical lymph node
metastasis, %
Extrathyroidal invasion, %
Psammoma body, %
Calcification, %
BRAF mutation, %
Follow-up period, mo
Whole Cohort
(n ⴝ 71)
43.73 ⫾ 15.57
30
10.1 ⫾ 2.61
2.88 ⫾ 1.09
43
42
Disease-Free
(n ⴝ 41)
38.73 ⫾ 12.48
22
9.04 ⫾ 2.38
2.62 ⫾ 0.88
24
29
Active Disease
(n ⴝ 30)
50.57 ⫾ 16.94
40
11.35 ⫾ 2.34
3.27 ⫾ 1.24
69
60
Univariate
P Value
.002
.142
.000
.022
.000
.015
46
42
61
45
127.0 ⫾ 29.8
29
35
51
44
130.37 ⫾ 22.36
70
50
73
47
122.11 ⫾ 38.17
.001
.326
.083
.812
.317
Multivariate
Adjusted
P Valueb
NS
.002
NS
.024
.025
NS
Abbreviation: NS, nonsignificant.
a
The data are shown as mean ⫾ SD unless otherwise indicated.
b
Variables considered for logistic regression models were based on the findings of univariate analysis.
c
Expression levels are presented as ⫺⌬Ct (miRNA-U6).
d
The low-risk group is defined as those patients who were less than 45 years old and had stage I PTC and those patients who were 45 years or
more of age with stage I or II PTC, according to the American Joint Committee on Cancer criteria. The remaining patients were defined as the
high-risk group.
or recurrent PTC during the follow-up period), whereas
the remaining cases with undetectable stimulated Tg level
and negative recurrence/persistence evidence were classified into the disease-free group. Five patients revealed
stimulated nonsuppressed Tg levels (all less than 10 ng/
mL) when undergoing postoperative radioiodine therapy.
This group of patients all had undetectable Tg under T4
suppression therapy in the absence of anti-Tg antibody
and negative image results during follow-up. Hence, these
5 patients were deemed as disease-free in our study. The
clinical characteristics of the whole cohort, the diseasefree group (n ⫽ 41), and active disease group (n ⫽ 30) are
presented in Table 1.
To determine whether miR-146b expression levels
were associated with poor prognosis, we analyzed the traditional survival risk factors and miR-146b levels of these
two groups. As shown in Table 1, advanced tumor stage,
presence of cervical lymph node (LN) metastasis, and
miR-146b expression were independent risk factors for
poor prognosis in PTC after adjusting variables based on
the findings of univariate analysis.
Figure 1 demonstrates the relationship between miR146b expression levels, the tumor stage (high risk vs low
risk), the presence of cervical LN metastasis (yes vs no),
and BRAF mutation (mutant vs wild-type) with the disease-free survival rate using Kaplan-Meier analyses. Patients with primary tumors expressing higher miR-146b
levels had a lower disease-free survival rate than those with
lower miR-146b expression levels (P ⬍ .05; Figure 1A).
Patients with advanced tumor stage or presence of cervical
LN metastasis revealed poorer disease-free survival rates
compared with those with lower tumor stage or absence of
cervical LN metastasis (P ⬍ .05; Figure 1, B and C). However, there were no significant differences between
patients harboring a BRAF mutation and control (ie, wildtype) in the disease-free survival rate (Figure 1D). Furthermore, high miR-146b expression correlated with poor disease-free survival rate in 31 PTCs with BRAF mutation
(P ⬍ .05) as shown in Supplemental Figure 1 (published on
The Endocrine Society’s Journals Online web site at
http://jcem.endojournals.org).
Additionally, the Cox proportional hazards model
analyses revealed that miR-146b expression was a significant prognostic factor for disease-free survival rate in patients with PTC (hazard ratio [HR] ⫽ 3.92; 95% confidence interval [CI], 1.73– 8.86). Advanced tumor stages
and cervical LN metastasis were poor prognostic factors
of disease-free survival in patients with PTC at follow-up
(HR ⫽ 3.98 [95% CI, 1.81– 8.79] and HR ⫽ 2.30 [95%
CI, 1.10 – 4.79], respectively).
High expression of miR-146b in BCPAP cells
generates significantly more cell colonies
and develops greater resistance to
chemotherapy-induced apoptosis
To evaluate the functional activity of miR-146b in
PTC, we augmented its expression in BCPAP cells by transiently transfecting them with a mimic-miR-146b, a double-stranded RNA oligonucleotide that was chemically
modified with Dharmacon ON-TARGET modifications.
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MiR-146b and Tumor Aggressiveness in PTC
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Figure 1. Association of miR-146b with disease-free survival rate, as shown by the Kaplan-Meier analysis of 71 PTC patients stratified as follows:
A, median of miR-146b expression, with high miR-146b expression correlated with poor disease-free survival rate (HR ⫽ 3.92 [95% CI,
1.73– 8.86]); B, high and low cancer risk group, with patients with advanced tumor stage correlated with poor disease-free survival rate (HR ⫽
3.98 [95% CI, 1.81– 8.79]); C) presence of cervical lymph node metastasis, with patients with cervical lymph node metastasis correlated with poor
disease-free survival rate (HR ⫽ 2.30 [95% CI, 1.10 – 4.79]; D, presence of BRAF mutation, with no significant disease-free survival rate difference
between BRAF mutation and wild-type group.
The expression of miR-146b in BCPAP cells was significantly higher after mimic-miR-146b transfection than
transfection with a nonspecific control (Figure 2A).
The oncogenic potential of miR-146b in PTC was evaluated via an in vitro colony-forming assay. BCPAP cells
transfected with a mimic-miR-146b had significantly
more colony foci compared with those transfected with a
nonspecific control (Figure 2, B and C). Both the growth
rate and sensitivity to chemotherapeutic drug-induced apoptosis were evaluated to determine the mechanism(s) responsible for the higher number of colonies formed by
BCPAP cells overexpressing miR-146b.
When we tested the influence of miR-146b on cell viability and proliferation by XTT assay, it was found that
BCPAP cells overexpressing miR-146b were significantly
less sensitive to cell-cell contact-induced growth inhibition
than those transfected with a negative control (Figure 3A).
Furthermore, these cells were treated with cyclophosphamide after transfection with either a mimic-miR-146b or
nonspecific control to determine their susceptibility to apoptosis. Transfection of BCPAP cells with miR-146b decreased the susceptibility of these cells to cyclophosphamide-induced apoptosis (Figure 3, B and C).
MiR-146b significantly increased cell migration and
invasion ability
In vitro gain-of-function experiments were performed
by transfecting BCPAP cells with a mimic-miR-146b and
negative control to determine whether miR-146b overexpression promotes tumor aggressiveness. As shown in Fig-
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pression of miR-146b may contribute to PTC
cell migration independently from BRAF
mutation.
Discussion
In the present study, it was found that miR146b expression levels in tumor tissue have a
prognostic value in patients with PTC at follow-up. Importantly, aside from the traditional clinical prognostic factors (ie, age at diagnosis, presence of extrathyroidal invasion,
and tumor stage), miR-146b is also a significant predictive factor of the outcome of PTC
patients. We have previously shown that miR146b is significantly associated with the tumor
stage and extrathyroidal invasion at the time of
surgery (14). In this study, we further demonstrate that miR-146b had a robust prognostic
value in PTC patients after a long-term followup. Thus, we report for the first time the prognostic implications of a single deregulated
miRNA in PTC. Furthermore, our in vitro
gain-of-function experiments suggested that
the overexpression of miR-146b enhances the
aggressiveness of PTC. Thus, the present study
not only highlights the functionality of miR146b in regard to regulating the invasiveness
and predicting the prognosis of PTC at follow-up but also offers evidence for the mechanistic basis of miR-146b in PTC.
Controversy exists about the prognostic
Figure 2. A, Relative expression levels of miR-146b after transfection with mimicvalue of BRAF mutation in PTC cohort studies.
miR-146b (146b) and nonspecific control (CTL) in BCPAP cells. The expression level is
presented as ⫺⌬Ct (miRNA-U6). B, High expression of miR-146b in BCPAP cells
In a meta-analysis, BRAF mutation in PTC is
generates significantly more cell colonies. Comparison of colony formation and
closely associated with extrathyroidal extenquantitative analysis of colony count in BCPAP cells transfected with mimic-miRsion, lymph node metastasis, advanced tumor
146b (146b) and control (CTL). BCPAP cells were seeded on day 0 at 1000, 500, and
100 cells per well. Colonies larger than 50 cells were scored after 2 weeks of
stages, disease recurrence, and even patient
incubation at 37°C. C, Quantitative analysis of the colony number of BCPAP cells
mortality (20). Furthermore, BRAF mutation
transfected with mimic-miR-146b (146b) and control (CTL). Data are presented as
was
an independent prognostic factor for rethe means of two independent experiments, and bars represent the SDs. *, 146b vs
CTL: P ⬍ .05.
current and persistent disease (21–23). Some
molecular mechanisms determining BRAF
mutation-promoted progression and the agure 4, A and C, miR-146b overexpression promotes cell
gressiveness of PTC have been uncovered. These include
migration and invasion of BCPAP cells. An overexpression
of miR-146b resulted in statistically significantly higher the down-regulation of major tumor suppressor genes and
BCPAP cell migration and invasion rates compared with thyroid iodide-metabolizing genes and the up-regulation
control cells (P ⬍ .05; Figure 4, B and D), thereby con- of cancer-promoting molecules, such as vascular endothefirming the aggressive properties of miR-146b in PTC. lial growth factor, matrix metalloproteinases, nuclear
Although BCPAP cells contain the homozygous BRAF transcription factor-␬B, and c-Met (3). BRAF mutation
mutation, similar results were obtained in TPC-1 cells, has been demonstrated to enhance tumorigenic ability in
which harbor RET/PTC rearrangement but not BRAF mu- transgenic mice in the thyroid gland (24). Studies in rat
tation (Supplemental Figure 2), suggesting that overex- thyroid cell lines showed that BRAF mutation promoted
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MiR-146b and Tumor Aggressiveness in PTC
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Figure 3. High expression of miR-146b in BCPAP cells resulted in a higher proliferation rate and greater resistance to chemotherapy-induced apoptosis. A,
Effect of miR-146b on cell proliferation ability determined by XTT assay. B, Effect of miR-146b on chemotherapy-induced (cyclophosphamide 0.015 M) apoptosis
in BCPAP cells. Detection of apoptosis using the flow cytometry after annexin V-FITC/propidium iodide (PI) staining in BCPAP cells transfected with mimic-miR146b (146b) and vector control (CTL) in the presence or absence of cyclophosphamide. Dot plots represent flow cytometric analyses of annexin-V-FITC
fluorescence (x-axis) vs PI-fluorescence (y-axis). Quadrant 1 (upper left), late necrosis; quadrant 2 (upper right), late apoptosis; quadrant 3 (lower left), live cells;
quadrant 4 (lower right), early apoptosis. C, Percentage of late, early apoptotic, and viable cells in overexpressed miR-146b BCPAP cells and controls. Data are
presented as the means of two independent experiments, and bars represent the SDs. *, Overexpression of miR-146b had significantly higher apoptosis (late plus
early) resistance compared with vector control (P ⫽ .043). MiR-146b-overexpressing BCPAP cells had no significant apoptosis rate difference compared with
vector control in the absence of cyclophosphamide treatment.
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common oncogene plasmids (RET and RAS) in
the PCCL3 cell line led to up-regulation of
miR-221 and miR-181b. Geraldo et al (29) further demonstrated that transformation of
BRAF mutation significantly gave rise to elevation of the miR-146b level. These results suggest an upstream regulatory role of oncogenes
in miRNA deregulation. In this study, miR146b, but not BRAF mutation, is an independent prognostic factor of PTC patients at follow-up, consistent with our previous crosssectional observation (14). Because miR-146b
expression is closely related to BRAF mutation
(14, 15) and probably the downstream target
of BRAF in PTC (29), and our in vitro gainof-function experiments suggest that miR146b may contribute to tumor aggressiveness
in PTC harboring BRAF mutation, it is interesting to note that miR-146b also exhibits aggressive features in other malignancies without
harboring BRAF mutation (18) and BRAF mutation may occur in melanoma (30) and colon
cancer (31), which were not associated with
overexpression of miR-146b. Our findings will
be helpful in the stratification of BRAF-positive tumors to determine those with poor prognosis with higher accuracy, needed for clinical
decisions. It might be expected that further
clarification of the causal relationship between
miR-146b and BRAF mutation in PTC would
help to solve some controversy in this area.
The target genes regulated by miR-146b remain largely unknown, and little has been reported regarding the molecular mechanisms by
which miR-146b influences tumor aggressiveFigure 4. MiR-146b augments invasive potential in BCPAP cells. A and B,
ness in PTC. Recently, Geraldo et al (29) demComparison and quantitative analysis of the cell migratory abilities of BCPAP cells
onstrated that miR-146b regulates the signal
transfected with mimic-miR-146b (146b) and control (CTL) in the Transwell (Corning)
migration assay. C and D, Comparison and quantitative analysis of the cell invasion
transduction pathways of TGF-␤ by repressing
abilities of BCPAP cells transfected with mimic-miR-146b (146b) and CTL in the
SMAD4 in thyroid tumorigenesis. MiR-146b
Matrigel (BD Biosciences) invasion assay. Data are presented as the means of two
overexpression in PCCL3 cells was also found
independent experiments, and bars represent the SDs. *, 146b vs CTL: P ⬍ .05.
to significantly increase cell proliferation in the
absence of TSH and conferred resistance to
Matrigel invasion of thyroid cells (25). In BRAF mutationTGF-␤-mediated cell cycle arrest. Further efforts aimed to
harboring human PTC-derived cells, transient transfecidentify miR-146b-regulated genes associated with tumor
tion with small interfering RNA to knock down BRAFaggressiveness will eventually identify novel biomarkers
inhibited cell growth and proliferation (26). These data all
strongly support the idea that BRAF mutation not only that can be used to correlate with disease outcome.
The molecular work-up of thyroid nodules on fine-neecan initiate tumorigenesis of PTC but also is required to
dle
aspiration (FNA) cytology samples has been shown to
maintain and promote the progression of PTC. However,
two studies in Japan (27) and Korea (28) published after improve the accuracy of FNA biopsy and also help predict
the meta-analysis mentioned above had shown that the tumor aggressiveness or behavior (32). Shen et al (33) reBRAF mutation is not associated with poor prognosis in ported that a set of 4 miRNAs (miR-146b, -221, -187, and
PTC. Pallante et al (12) showed that transformation of -30d) were found to be able to differentiate malignant
E204
Chou et al
MiR-146b and Tumor Aggressiveness in PTC
from benign lesions in thyroid FNA specimens, as determined by quantitative PCR. Further studies are required to
demonstrate whether the expression level of miR-146b in
thyroid FNA biopsy specimens has potential utility as diagnostic and prognostic indicators in PTC, thus facilitating more accurate risk stratification and optimizing surgical and postsurgical management of patients with PTC.
Some limitations of this study should be noted. First, it
is a retrospective study with the selection bias and reporting bias inherent in all retrospective studies. Because of the
small number of patients analyzed in this study, further
studies on a great number of patients are required to confirm our findings. Second, the expression level of miR146b in this study is arbitrary. The cutoff level of miR146b in tumor tissue to predict prognosis remains to be
established. Finally, our in vitro gain-of-function experiments were performed in a PTC cell line containing BRAF
mutation. It has yet to be proven whether miR-146b exhibited similar manifestation in PTC cell lines without
BRAF mutation.
Conclusion
In the present study, the prognostic role and functionality of miR-146b in PTC were demonstrated. Our results
suggest that miR-146b is a novel prognostic molecular
marker of PTC, and the in vitro functional studies provide
a mechanistic explanation for miR-146b in determining
the aggressiveness of PTC. Identifying miR-146b-regulated genes associated with tumor aggressiveness will enhance our understanding of the molecular mechanisms
involved in tumor aggressiveness in PTC, provide new
prognostic biomarkers, and ultimately offer new leads for
developing therapies for PTC.
Acknowledgments
We thank Professor Jacques Dumont from Universite Libre de
Bruxelles for providing thyroid cancer cell line BCPAP.
Address all correspondence and requests for reprints to:
Hong-Yo Kang or Rue-Tsuan Liu, Division of Metabolism, Department of Internal Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center, 123 Ta-Pei Road, Niao-sung
District, Kaohsiung City, Taiwan 833. E-mail: ruetsuan@ms2.
hinet.net or [email protected].
This work was supported by the following grants: National
Science Council of the Republic of China, Taiwan Grants: NSC
98-2314-B-182A-014-MY2 (to R.-T.L.); NSC 100-2314-B182A-06 (to C.-K.C.).
Disclosure Summary: There are no conflicts of interest to
disclose.
J Clin Endocrinol Metab, February 2013, 98(2):E196 –E205
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