11

Down-regulation of microRNAs controlling tumourigenic

factors in follicular thyroid carcinoma
Maria Rossing1,2, Rehannah Borup1, Ricardo Henao5, Ole Winther5, Jonas Vikesaa1,
Omid Niazi1, Christian Godballe3, Annelise Krogdahl4, Martin Glud1,
Christian Hjort-Srensen3, Katalin Kiss6, Finn Noe Bennedbk2* and Finn Cilius Nielsen1*
1
Center for Genomic Medicine, University of Copenhagen, Rigshospitalet, DK - 2100 Copenhagen, Denmark
2
Department of Endocrinology and Metabolism, Herlev Hospital, Copenhagen, Denmark
3
Department of ENT Head and Neck Surgery, Odense, Denmark
4
Department of Pathology, Odense University Hospital, Odense, Denmark
5
Bioinformatics Centre, University of Copenhagen, Copenhagen, Denmark
6
Department of Pathology, Gentofte Hospital, Gentofte, Denmark

(Correspondence should be addressed to F C Nielsen; Email: fcn@rh.dk)

*(F N Bennedbk and F C Nielsen contributed equally to this work)

Abstract
The molecular determinants of thyroid follicular nodules are incompletely understood and assessment of malignancy is
a diagnostic challenge. Since microRNA (miRNA) analyses could provide new leads to malignant progression,
we characterised the global miRNA expression in follicular adenoma (FA) and follicular carcinoma (FC). Comparison of
carcinoma and adenoma with normal thyroid revealed 150 and 107 differentially expressed miRNAs respectively. Most
miRNAs were down-regulated and especially miR-199b-5p and miR-144 which were essentially lost in the carcinomas.
Integration of the changed miRNAs with differentially expressed mRNAs demonstrated an enrichment of seed sites
among up-regulated transcripts encoding proteins implicated in thyroid tumourigenesis. This was substantiated by the
demonstration that pre-miR-199b reduced proliferation when added to cultured follicular thyroid carcinoma cells. The
down-regulated miRNAs in FC exhibited a substantial similarity with down-regulated miRNAs in anaplastic carcinoma
(AC) and by gene set enrichment analysis, we observed a significant identity between target mRNAs in FC and
transcripts up-regulated in AC. To examine the diagnostic potential of miRNA expression pattern in distinguishing
malignant from benign nodules we employed a supervised learning algorithm and leave-one-out-cross-validation. By this
procedure, FA and FC were identified with a negative predicted value of 83% (data generated by microarray platform) and
of 92% (data generated by qRT-PCR platform). We conclude that follicular neoplasia is associated with major changes in
miRNA expression that may promote malignant transformation by increasing the expression of transcripts encoding
tumourigenic factors. Moreover, miRNA profiling may facilitate the diagnosis of carcinoma vs adenoma.
Journal of Molecular Endocrinology (2012) 48, 1123

Introduction As the latter features may be overlooked it is generally

Thyroid nodules are found in up to 7% of the adult
population (Hegedus et al. 2003). Although the
majority of the nodules are benign, carcinoma of
the thyroid gland is the most common malignancy of
the endocrine system (Curado & Edwards 2007).
Follicular adenomas (FA) are benign, encapsulated
tumours and they are five times more frequent than
follicular carcinomas (FC; Faquin 2008). FC represent
1015% of all thyroid malignancies and mainly occur in
middle-aged euthyroid women as a painless thyroid
nodule (Faquin 2008). FA and FC are morphologically
similar and carcinomas are distinguished by vascular
and/or capsular invasion (Schmid & Farid 2006).
Journal of Molecular Endocrinology (2012) 48, 1123
09525041/12/048011 q 2012 Society for Endocrinology
accepted, that application of biomarkers could improve
diagnosis (Franc et al. 2003).
MicroRNAs (miRNAs) are non-coding single-
stranded RNAs of about 22 nucleotides. miRNAs
regulate translation and stability of particular target
mRNAs by imperfect base pairing (Bartel 2004). It is
estimated that the number of miRNAs may exceed 1000
(http://microrna.sanger.ac.uk/) and miRNAs regulate
about one-third of the mammalian protein-coding
mRNAs (Bartel 2009, Friedman et al. 2009). The
expression of miRNAs is temporally and spatially
regulated. Many are important for the differentiation
processes during particular developmental stages,
but miRNAs also exhibit important functions in mature
DOI: 10.1530/JME-11-0039
Printed in Great Britain Online version via http://www.endocrinology-journals.org
12 M ROSSING, F N BENNEDBK, F C NIELSEN and others . miRNAs in follicular thyroid tumours

cells (Schmittgen 2008). Moreover, miRNAs are data indicated that miRNAs could promote malignant
aberrantly expressed or lost in a variety of cancers progression, we further employed classification algo-
(Rosenfeld et al. 2008). Many target mRNAs encode rithms to distinguish carcinoma from adenoma.
oncogenes and tumour suppressors and in this way
dysregulated miRNAs may play a causal role in
malignant progression. Not surprisingly, miRNAs are
therefore considered attractive candidates for classi- Materials and methods
fication of tumours. The role of miRNAs in thyroid
cancer is incompletely understood. A number of Thyroid tissue, FA and FC
miRNAs have previously been identified in various Thyroid samples originated from a consecutive series
thyroid tumours (He et al. 2005, Pallante et al. 2006, of patients with a solitary or prominent scintigraphically
Weber et al. 2006, Chen et al. 2008, Nikiforova et al. cold thyroid nodule operated on, and with a histological
2008). miR-197 and miR-346 are overexpressed in FC in diagnosis of an FA or FC. As a uniform histopathological
comparison to adenoma and in vitro studies suggested evaluation was essential, the diagnosis was made by a
that both miRNAs could have a significant impact on particular pathologist specialising in thyroid pathology.
tumour cell proliferation (Weber et al. 2006). All tumours were diagnosed and classified according to
In this study, we employed global miRNA analysis the WHO definition of histological criteria. Clinical data
to identify differentially expressed miRNAs in follicular are listed in Table 1. Surgically removed thyroid samples
thyroid carcinoma and adenoma. miRNAs were inte- were snap frozen at the Department of Pathology and
grated with differentially expressed mRNAs to identify stored at K120 8C over a 5-year period. The project was
and validate putative target mRNAs. The gene ontology approved by the ethics committee and informed consent
and gene set enrichment analysis (GSEA) of these was obtained from all patients. Twelve FA, twelve FC and
showed a significant enrichment of seed sites among ten normal thyroid (NT) specimens were included.
transcripts encoding proteins involved in thyroid NT specimens were obtained by a thyroid pathologist to
tumourigenesis and an overlap to transcripts up-regu- ensure that the tissue derived from macro- and micro-
lated in anaplastic carcinoma (AC) respectively. As the scopically normal tissue adjacent to the encapsulated
Table 1 Clinical data of patients with thyroid follicular neoplasia. Twelve patients with histopathologically verified follicular carcinomas (FC),
minimal and widely invasiveness, and twelve patients with follicular adenomas (FA). The table depicts diagnosis, age, sex, tumour size,
invasiveness of the examined tumours and status of known oncogenes. All tumours were negative for KRAS point mutation and examination of
BRAF showed only one positive carcinoma sample positive for BRAF point mutation; K601E (c.1801AOG p.Lys601Glu). Examination for
PAX8/PPARg translocation exhibited no positive samples

Nodule size Invasiveness BRAF

Diagnosis Age (years) Sex (M/F) (cm; max diameter) (minimal vs widely) gene mutation

FC_01 32 F 6 Minimal No mutation

FC_02 60 F 2.5 Minimal* No mutation
FC_03 32 F 1.5 Minimal No mutation
FC_04 48 F 5 Widely No mutation
FC_05 41 M 6 Minimal No mutation
FC_06 75 F 4 Minimal* No mutation
FC_07 35 M 7 Minimal No mutation
FC_08 59 F 2 Widely No mutation
FC_09 28 M 2.5 Minimal No mutation
FC_10 46 F 10.5 Widely No mutation
FC_11 32 F 4 Minimal Mutation
FC_12 63 F 3.5 Minimal No mutation
FA_01 55 F 3.5 No mutation
FA_02 41 M 4 No mutation
FA_03 65 F 4 No mutation
FA_04 47 F 2.5 No mutation
FA_05 63 F 4.5 No mutation
FA_06 33 F 2 No mutation
FA_07 37 F 3.5 No mutation
FA_08 46 M 3 No mutation
FA_09 37 F 2.5 No mutation
FA_10 51 F 4 No mutation
FA_11 65 F 4 No mutation
FA_12 37 F 4 No mutation
*indicates the two FC (FC_02 and FC_06) of the oncocytic type.

Journal of Molecular Endocrinology (2012) 48, 1123 www.endocrinology-journals.org

 miRNAs in follicular thyroid tumours .
tumours. The number of tumours was balanced to
provide optimal power estimates and a similar number
of samples in each diagnostic category.
Total RNA and DNA isolation
Total RNA was isolated from frozen samples using
Trizol (Invitrogen) according to the manufacturers
protocol. RNA was precipitated using 100% iso-
propanol. Purified RNA was subsequently quantified
on a NanoDrop ND-1000 Spectrophotometer (Nano-
Drop Technologies, Wilmington, DE, USA) and
examined on a Bioanalyzer Nano RNA Chip (Agilent
Technologies, Santa Clara, CA, USA) before labelling.
Each sample (1 mg) was pooled and used as common
reference. DNA from tissues was isolated by lysing the
tissue in 20 ml proteinase K and 200 ml Tris/NaCl/
EDTA/SDS, followed by overnight incubation at 55 8C.
Finally, 5 M NaCl is added to the lysed tissues and DNA
is precipitated by adding 200 ml ice-cold 96% ethanol.
Total RNA for mRNA microarray was extracted from
pre-miRNA transfected cells using NucleoSpin RNA/
Protein from Macherey-Nagel according to their
recommendations.
Detection of point mutations
Mutation analyses, including PCR-amplification and
sequencing, were performed on all 24 tumour samples
for KRAS and BRAF (V600E and K601E) and regular
PCR-amplification, followed by gel-separation was used
to analyse the fusion point of PAX8/PPARg transloca-
tion. For detection of possible amplicons, a positive
control was examined along with the 24 tumour
samples. Primer sequences for KRAS, forward;
5 0 -TGTAAAACGACGGCCAGTCGATACACGTCTGC-
AGTCAA-3 0 and reverse; 5 0 -CAGGAAACAGCTATG-
ACCCTCATGAAAATGGTCAGAGA-3 0 , BRAF, forward;
5 0 -TGCTTGCTCTGATAGGAAAATG-3 0 and reverse;
5 0 -GGATGGTAAGAATTGAGGCT-3 0 and PAX8/PPARg,
forward; 5 0 -GCATTGACTCACAGAGCAGCA-3 0 and
reverse; 5 0 -AGACCACTCCCACTCCTTTG-3 0 (Kroll
et al. 2000).
miRNA and mRNA expression analyses
miRNA expression levels were determined by micro-
array analysis. Total RNA (1 mg) was labelled with
fluorescent Hy3 (sample)/Hy5 (reference-sample)
dye from the miRCURY LNA microRNA Array Power
Labelling Kit (Exiqon, Vedbaek, Denmark) in accord-
ance with the manufacturers instructions. Using a
TECAN hybridisation station, labelled samples were
hybridised overnight to pre-printed miRCURY LNA
microRNA Array, v.11.0 (Exiqon), containing probes
www.endocrinology-journals.org
M ROSSING, F N BENNEDBK, F C NIELSEN and others 13
for 841 human miRNAs, catalogued in the miRBase
Sequence Database (Release 11.0; http://microrna.
sanger.ac.uk/) and 428 proprietary human miRPlus
sequences not yet annotated in miRBase.
Furthermore, miRNA expression levels from 30
thyroid specimens (12 FC, 12 FA and 6 NT), were
generated using the miRCURY LNATM Universal RT
miRNA PCR panels I and II, V2 (Exiqon). Total RNA
(40 ng) was reverse transcribed using the Universal
cDNA synthesis kit, mixed with SYBR Green master
mix kit, and subsequently added to the pre-aliquoted
miRNA PCR primer sets in two 384-well PCR plates
enabling profiling of 742 human miRNAs. All reagents
were from Exiqon and their recommendations were
followed. Each plate contained an additional six primer
sets for reference miRNAs and a set of negative
controls. The amplification curves were analysed using
the Roche LC software, both for determination of
crossover point (Cp) and for melting curve analysis.
One hundred and thirty-five miRNA assays were
successfully assessed with sufficient signal (Cp !37, or
5 Cp less than negative controls) in all samples.
Analyses of global mRNA levels in RNA extracted
from pre-miRNA transfected cells were performed
by the Affymetrix platform. Total RNA (250 ng) was
amplified and labelled using the Ambion WT
Expression Kit (Applied Biosystems) according to the
manufacturers instructions. The labelled samples were
hybridised to the Human Gene 1.0 ST GeneChip array
(Affymetrix, Santa Clara, CA, USA). Arrays were washed
and stained with phycoerythrin-conjugated streptavidin
(SAPE) using the Affymetrix Fluidics Station 450,
and subsequently scanned in the Affymetrix GeneArray
2500 scanner to generate fluorescent images, according
to the Affymetrix GeneChip protocol. Cell intensity
files (CEL files) were generated in the GeneChip
Command Console Software (AGCC; Affymetrix).
CEL files were modelled using the robust multichip
average approach, followed by mean Probe Set
Summarisation resulting in a single expression value
for each gene. Modelled CEL files were generated with
the software package Partek Genomics Suite 6.5.
Image analysis and normalisation of miRNA
expression
Arrays were scanned in an Agilent DNA Microarray
Scanner (Agilent Technologies) and resulting images
were analysed with Genepix Pro 6.0 software (Molecular
Devices). Background intensities were subtracted
from foreground intensities and within array LOESS
normalised, followed by Aquantile normalisation
between arrays as implemented in the Limma package
in Bioconducter library (Gentleman et al. 2004) in
R version 2.10 (R Development Core Team 2007). Mean
values of the quadruplicate probes for each of the
Journal of Molecular Endocrinology (2012) 48, 1123
14 M ROSSING, F N BENNEDBK, F C NIELSEN and others . miRNAs in follicular thyroid tumours
miRNAs were obtained and log2 ratios between sample
and reference were used for further analyses.
Normalisation of the results derived from the RT
miRNA PCR panels was performed based on the average
of the assays detected in all samples; Normalised
CpZaverage samples Cp (nZ135)Kassay Cp. The
normalised miRNA expression values were used for
generating a diagnostic classifier between FC and FA
as described in the section Construction of classifier.
Class comparison analyses
Class comparison was performed by the Limma package
(http://bioconductor.org/packages/release/bioc/
html/limma.html). For each miRNA standard errors
and fold changes were estimated by fitting a linear
model. Differentially expressed miRNAs were
determined by applying empirical Bayes moderated t-
statistics test (Wettenhall & Smyth 2004). miRNAs were
defined to be differentially expressed if they had a
BenjaminiHochberg corrected P value below 0.05 and
an absolute fold-change above 1.5.
mRNA expression data and miRNA target predictions
followed by pathway analysis
The global mRNA expression data originating from
FA, FC and AC tissue samples was based on previous
work by our group (Borup et al. 2010). In addition,
mRNA profiles of papillary and NT tissue samples were
down loaded from the Array Express, ID: E-GEOD-6004
and E-GEOD-7307 respectively. To predict mRNA
targets, based on the observed miRNA profiles, we
implemented the method developed by Gallagher et al.
(2010). Briefly, mRNA target predictions were based
on the TargetScan miRNA target prediction database
in combination with the observed changes in miRNAs.
Only miRNAs that exhibited an absolute change
O1.5-fold and mRNA with an average expression
intensity O40 in FCs were included in the analysis.
This method provides a weighted miRNA inhibitor
score (sum of effects), predicting the transcripts, most
likely to be regulated by miRNAs. As the majority of the
differentially expressed miRNAs were down-regulated,
only mRNAs with a positive composite score were
considered in the analysis. A cutoff at C2 and a variance
filtering of 0.176 were used to remove unhybridised
probes in our weighted inhibitor score list (composite
score). This resulted in the retrieval of 1381 probe sets,
representing 528 genes. To examine whether the
putative mRNA targets were associated with particular
biological functions or pathways, the predicted mRNAs
were examined in the Ingenuity Pathway Analysis soft-
ware package (Ingenuity Systems, www.ingeuity.com).
The most significant pathways were identified by the
Journal of Molecular Endocrinology (2012) 48, 1123
Fishers exact test. GSEA of the target list was submitted to
and performed using the open source software available
at http://www.broadinstitute.org/gsea/msigdb/index.
jsp. The gene list was loaded into the molecular signature
database (MSigDB) and overlaps were detected in the
C2 currated gene analysis. To determine the association
to the gene set, the hypergeometric distribution of
overlapping genes over all genes in a gene set was
calculated. For this analysis a cutoff was set at composite
score OC5 and only considering the differentially
expressed genes, a cutoff was set at P!0.01, qZ0.0036,
resulting in 287 probes. Heatmaps were generated as
supervised (two-way) cluster analysis.
Construction of classifier
Normalised data from 24 samples consisting of the
expression level of 545 different probes (377 annotated
miRNAs and 168 hsa miRPlus, all with an A value O7,
AZ12 (log2RedClog2Green)) were included in the
analysis. The classifier was constructed to classify FC
and FA. The validation of the classifiers performance
on the training set was done by leave-one-out-cross-
validation (LOOCV), that provides an unbiased
estimate of the prediction error by going over all
training examples in turn using the complement
training set (of size 24K1Z23) to perform training
(including probe ranking, selection and model fitting)
and using the last left out sample for prediction. In this
way, we obtained as many predictions as there were
samples in the training set. These predictions formed
unbiased estimates of the prediction error quantified in
terms of the confusion matrix. The training of the
classifiers inside the LOO loop consisted of a univariate
selection step followed by applying support vector
machine learning (SVM; Vapnik 1998). Probes were
ranked according to their differential expression by
the Students t-test with a threshold of P!0.001
(Dudoit et al. 2003). A permutation test was performed
to determine if the cross-validated misclassification
rate is lower than expected by chance (Tusher et al.
2001, Simon et al. 2007). In 1000 random permutations
of the class label, the entire cross-validation was
repeated for classifying the random classes of samples.
The proportion of the 1000 random permutations, that
gives a smaller or similar cross-validation misclassifi-
cation rate as obtained with the real data, determined
the permutation P value. The statistical significance of
the error rate was determined for the SVM classifier.
Quantitative reverse transcription PCR of miRNA
and mRNA
cDNA was prepared from 25 ng total RNA from
34 tumour samples using TagMan microRNA reverse
www.endocrinology-journals.org
 miRNAs in follicular thyroid tumours .
transcription kit and TagMan microRNA assays
containing predesigned primers for miR-221, miR-
182, miR-96, miR-199a3p, miR-144*, miR-199b5p and
miR-1826 was added. MiR-191 was used for endogenous
control. Quantitative reverse transcription PCR (qRT-
PCR) was performed by TagMan universal PCR master
mix No AmpEras UNG, according to the manufac-
turers instructions, all from Applied Biosystems. Each
amplification reaction was performed in triplicate, and
median value of the three cycle threshold was used for
further analyses. For calculations of fold changes we
used the 2KDCt method (Schmittgen & Livak 2008). In
addition, we validated Exiqon miRPlus probe, miRPlus-
E-1078, used for classification of FC and FA. For this
qRT-PCR analysis we employed the Exiqon microRNA
LNA PCR primer sets together with Universal cDNA
synthesis and SYBR green master mix, following the
manufacturers instructions (Exiqon).
For validation of miRNA predicted transcripts 2 mg
total RNA from 24 tumour samples (12 FC and 10 NT)
was reverse transcribed to cDNA using High-Capacity
cDNA Reverse Transcription kit (Applied Biosystems).
Synthesised cDNA (200 ng/ml) diluted 1/100-fold and
2 ml aliquot used in 10 ml total PCR containing fast
SYBR green PCR Master Mix (Applied Biosystems) and
10 pmol/ml of each primer for target genes. Primer
sequences for VPS26A, forward; 5 0 -GCTTGCCACC-
TATCCTGATG-3 0 and reverse; 5 0 -CTGGGTCCAATTC-
CTGTGAT-3 0 and ABCC1, forward; 5 0 -TAAAAGAGG-
ATGCCCAGGTG-3 0 and reverse; 5 0 -ACCCTGTGATCC-
ACCAGAAG-3 0 . b-Actin was measured for endogenous
control. The PCR comprised 40 cycles of a four-step
programme: step 1, 50 8C for 2 min, step 2, 95 8C for
2 min, step 3 (40 cycles), 95 8C for 10 s and 60 8C for
45 s and step 4, 95 8C for 15 s, 60 8C for 15 s and 95 8C
for 15 s. qPCRs were performed in triplicate.
miRNA transfections and proliferation experiments
Human thyroid follicular R082 W-1 cancer cells were
grown at 37 8C, 5% CO2 in RPMI 1640 with GlutaMax
(Invitrogen) supplemented with 10% FBS (Invitrogen),
1% penicillin and streptomycin. Cells were trans-
fected with 50 nM pre-miR-199b and pre-miR-144,
both from Applied Biosystems and for control
miRNA, we used miRIDIAN microRNA mimic negative
control#2 (Dharmacon/Thermo Scientific, hafayette,
CO, USA). Cells were transfected with Turbofect
transfection reagent (Dharmacon/Thermo Scientific)
according to the manufacturers instructions. Cell
proliferation was studied real-time using the XCELLi-
gence system as described in the manufacturers
instructions (Roche Applied Science and ACEA
Bioscience). This allowed continuous measurements
of the electronic impedance by detecting the physio-
logical changes of the cells on the electrodes attached
www.endocrinology-journals.org
M ROSSING, F N BENNEDBK, F C NIELSEN and others 15
to the E-96. Five hours post-transfection with pre-
miRNAs, transfected cells were plated in quadruplicates
and monitored for 5 days. The experiment was repeated
three times. For mRNA microarray analysis, total RNA
was extracted 24 h after pre-miRNA transfections and
labelled as described in the section miRNA and mRNA
expression analyses.
Results
Tumour samples and oncogenic mutations
The thyroid samples originated from a consecutive
series of patients and included tumours from 19 women
and 5 men and all tumours were classified according
to the WHO definition of histological criteria. The
median age was 44 years in carcinoma patients and 47
years in adenoma patients. The size of the tumours
ranged from 1.5 to 10.5 cm and the median diameter
was 4 cm in the carcinoma patients and 3.75 cm in the
adenoma patients (Table 1). All tumours were
examined for KRAS and BRAF mutations and this
showed that only one carcinoma sample was positive for
BRAF and examination for PAX8/PPARg translocation
with PCR followed by gel-separation showed no positive
amplicons (Table 1). Taken together, the thyroid
specimens in the two diagnostic groups were com-
parable both with respect to the clinical features and
the presence of oncogenic mutations.
miRNA expression in FC and FA
The following class comparison analysis and miRNA
target analysis are based on the derived microarray
expression data since this platform counts the largest
number of miRNAs. Class comparison analysis
revealed 150 annotated and differentially expressed
human miRNAs 37 up-regulated and 113 down-
regulated miRNAs in FCs compared with NT.
The fold change ranged from 3.1- to K39-fold.
Owing to the substantial 39-fold down-regulation of
miR-199b-5p (also named miR-199b); this miRNA
is essentially lost in FC. MiR-144*, miR-199b-3p, miR-
199a-5p and miR-144 were also strongly reduced
almost to the background. Of the most up-regulated
miRNAs, miR-221, miR-96 and miR-182 exhibited
fold changes of 3.1, 2.9 and 2.6 respectively. The
comparison of FA to NT tissue revealed 107
differentially expressed miRNAs. Forty-two were
up-regulated and 65 were down-regulated. Finally,
the comparison of carcinoma to adenoma showed
that 56 miRNAs were differentially expressed between
these groups. Twelve miRNAs were up-regulated
and 44 were down-regulated in the carcinoma. The
five most up- and down-regulated miRNAs in each
Journal of Molecular Endocrinology (2012) 48, 1123
16 M ROSSING, F N BENNEDBK, F C NIELSEN and others . miRNAs in follicular thyroid tumours

Table 2 Dysregulated miRNAs. The five most up- and down- Computational identification of putative target
regulated miRNAs in the three comparisons, follicular carcinomas mRNAs and pathway analysis
(FC) vs normal thyroid (NT), follicular adenomas (FA) vs NT
and FC vs FA are listed. miRNAs were defined to be differentially Results from the global expression profiling of NT and
expressed if they had a BenjaminiHochberg corrected P value FA and FC samples were used for integration of mRNA
below 0.05 and an absolute fold-change above 1.5
and miRNA array data. In a pilot analysis, we counted
Fold Adjusted predicted seed sites corresponding to the changed
miRNA change P value P value miRNAs from the differentially expressed transcripts
using the Targetscan miRNAmRNA integration soft-
FC vs NT ware in Partek Genomic Suite (http://www.partek.
miR-199b-5p K39 3.15!10K31 5.98!10K28 com/partekgs). Based on the fact that the vast majority
miR-144* K15.1 7.10!10K9 2.50!10K7 of mRNAs were down-regulated by associated miRNAs
miR-199b-3p K12.1 1.33!10K28 1.26!10K25
(Guo et al. 2010), only mRNAs and miRNAs that
miR-199a-5p K9 1.66!10K14 1.97!10K12
miR-144 K6.9 3.34!10K21 1.06!10K18 exhibited an inverse expression pattern were
miR-221 3.1 1.12!10K5 0.0001 considered. We detected a significant enrichment
miR-96 2.9 5.92!10K6 8.72!10K5 (Fishers exact test P!0.01, assuming that the total
miR-182 2.6 2.11!10K6 3.46!10K5 number of 904 miRNA could target 22.000 transcripts
miR-597 2.4 0.002 0.0114
miR-222 2.3 0.0007 0.0049 at a global level) of seed sites corresponding to down-
FA vs NT regulated miRNAs. The down-regulated miRNAs in
miR-199b-5p K26 4.69!10K27 8.92!10K24 the FC group exhibited putative seed sites in almost
miR-144* K8.1 6.76!10K6 0.0001 85% of the up-regulated transcripts, which distin-
miR-663 K6.2 5.48!10K12 4.51!10K10 guished carcinoma from NT and adenoma respectively.
miR-199b-3p K6.1 1.26!10K18 4.80!10K16
miR-142-3p K4.6 2.34!10K15 4.46!10K13 All limitations of the computational approach taken
ebv-miR-BART8 5.3 0.0083 0.0489 into consideration, the results suggest that the changed
miR-517a 4.5 0.0043 0.0285 miRNAs could have an impact on the observed changes
miR-512-3p 3.6 1.70!10K3 1.37!10K2 in the transcriptome during progression to carcinoma.
miR-301b 3.2 5.08!10K6 8.55!10K5
miR-518a-3p 2.60 6.36!10K3 3.94!10K2
To identify the biological pathways and further
substantiate the possibility that miRNA changes had an
FC vs FA
miR-512-3p K3.2 0.0032 0.0395 impact on the gene expression pattern in the carcinoma,
miR-886-5p K3 2.58!10K6 0.0003 we employed the previously reported ranking system
miR-450a K3 6.92!10K7 0.0001 for mRNA target identification (Gallagher et al. 2010).
miR-301b K2.6 5.78!10K5 0.0032 Following miRNA target identification, 528 of the
miR-429 K2.4 0.0001 0.0046
miR-637 2.1 7.09!10K5 0.0036 highest ranked transcripts with composite score OC2,
miR-631 2 0.0028 0.0364 was loaded into the geneontology and pathway analysis
miR-219-2-3p 1.8 0.0041 0.0467 feature of the Ingenuity Software. Almost 200 of the
miR-662 1.8 0.0017 0.0272 putative targets mRNAs encoded factors categorised in
miR-744 1.8 2.21!10K7 7.00!10K5
neoplasia, cancer and tumourigenesis (P values of
1.95!10K5, 2.16!10K5 and 2.25!10K5 respectively).
comparison are listed in Table 2 and the complete When we explored the biological functions, a group
list of miRNAs that changed more than twofold is of 17 transcripts (APC, ATM, CCDC6, CCND1, COPS2,
listed in Supplementary Table 1 (see section on ETS1, FN1, GABBR2, LIFR, NRAS, PPARGC1A, PTPN4,
supplementary data given at the end of this article). RAP2A, S1PR1, SMAD2, TGFBR1 and VEGFA; PZ4.42
Fifty-eight miRNAs were differentially expressed in !10K5) encoded factors previously described in thyroid
both the carcinoma and adenoma compared with cancer. Corresponding to the down-regulation of miR-
NT (Fig. 1A). The overlap was marked among the NAs, 9 out of 11 target transcripts within the thyroid
down-regulated miRNAs, where 49 of the 65 down- cancer specific mRNAs were increased (82%; P!0.05).
regulated miRNAs in the adenoma were identical, To further examine the putative biological signi-
compared with 9 out of 42 for the up-regulated ficance of miRNA changes, we performed a GSEA
miRNAs. With the exception of miR-199b-5p, that (http://www.broadinstitute.org/gsea/msigdb/index.
were progressively down-regulated in carcinoma, jsp). The target list was filtered by a composite score
the shared miRNAs exhibited basically the same OC5 and only differentially expressed genes were
levels in adenoma and carcinoma compared with NT included (P!0.01, qZ0.0036) in the analysis, as shown
(Fig. 1B). Taken together, the results imply that in Fig. 2B. This resulted in 287 probe sets of which 260
changes in miRNA expression predominantly occur probe sets were up-regulated. The 260 probes corre-
during transition from NT epithelial cell to adenoma sponded to 135 different genes, which were uploaded
and not during malignant transformation. into GSEA and underwent a C2 currated genes analysis.
Journal of Molecular Endocrinology (2012) 48, 1123 www.endocrinology-journals.org

A Proliferative effects of selected miRNAs and
FC FA
92 58 49
28 9 33
64 49 16
B control. Whereas, the doubling time based on the
4
miRNA down-regulation (fold)
10
miRNA up-regulation (fold)
3
0
10
2 20
30
1 199b-5p and examined the genome-wide transcriptome
40
0 50
NT FA FC
Figure 1 miRNA expression in follicular carcinoma (FC) and
adenoma (FA). (A) Venn diagram showing differential and common
miRNAs among FC and FA in relation to normal thyroid (NT) tissue.
The total number of differentially expressed miRNAs is shown in
black, and the number of up- and down-regulated miRNAs is
shown in green and red respectively. (B) The graphs show the
fold change of 9 common up-regulated (green) and 49 common
down-regulated (red) miRNAs, respectively, in relation to NT.
The Rodrigues_thyroid_carcinoma_anaplastic_up-
regulated_cluster that contains 17 (82 probes) of our
135 genes was significantly connected to our target
list (PZ1.55!10K6). The 17 overlapping transcripts
are GSK3B, FXR1, WAPAL, ITCH, NUPL1, SLC7A11,
DDX3X, KPNA4, STK4, MAP3K7IP3, SSX2IP, TBL1XR1,
SIX4, EIF5B, PRPF40A, NARG1 and USP31. The relative
expression levels of the overlapping transcripts and of
the entire Rodrigues cluster in FC and NT are depicted
in heatmap C and D, Fig. 2. As shown in heatmap D,
Fig. 2, more than 90% of the transcripts included
in the Rodriques cluster were also up-regulated in FC.
The significance of the overlap between FC and AC
was further reinforced by a comparison of the recent
characterisation of miRNAs in AC (Braun et al. 2010),
since miR-199b, miR-100, miR-101, miR-31, miR-17,
miR-99a, miR-125b, miR-26a, miR-708, miR-486, let-7i
and let-7g, are down-regulated in both FC and AC.
The overlap between AC and FC indicated that these
cancers exhibited common features in their miRNA
expression patterns, and we therefore examined the
expression pattern of the 135 miRNA target transcripts
in FC, papillary carcinoma (PC) and AC, see heatmap
E, Fig. 2. In accordance with the GSEA, the 135
transcripts showed a unique expression pattern for FC
and AC as opposed to the PC that were clearly
distinguished by this gene set.
www.endocrinology-journals.org
miRNAs in follicular thyroid tumours . M ROSSING, F N BENNEDBK, F C NIELSEN and others 17
functional target validation
To examine the functional significance of the miR-
199b-5p and mir-144 that were strongly down-regulated
in FC, we determined their effect on proliferation of
thyroid carcinoma R082 W-1 cells. Cells were trans-
fected with pre-miR-199b and pre-miR-144 and a
miRIDIAN microRNA Mimic was included as negative
normalised cell index showed a significant w23%
(P!0.004) decrease upon pre-miR-199b-5p transfec-
tions, the effect of pre-miR-144 was only 8% (PZ0.056;
Fig. 3). The results showed that down-regulation of
miR-199b-5p, may be causally involved in the growth of
FC. To validate the computational predicted targets,
we subsequently transfected R082 W-1 cells with miR-
to identify transcripts that were regulated by the
miRNA. In total 581 transcripts were down-regulated
NT FA FC
and 733 were up-regulated. Out of the 190 compu-
tationally predicted miR-199b-5p targets 56 were
represented among the down-regulated mRNAs.
Among the up-regulated only seven transcripts were
predicted targets. Thus, down-regulated target mRNAs
were enriched in the list of computational predicted
target transcripts (Fishers exact test, P!0.0005),
whereas there was no enrichment (PZ0.34) among
up-regulated transcripts. Our data show that about
30% of the computational predicted targets are likely
to represent in vivo targets. We subsequently explored
the expression levels of the 56 transcripts in the FC.
Data were filtered by a P value of 0.05 resulting in 39
differentially expressed transcripts where 87% were
significantly up-regulated, corresponding to the loss of
199b-5p in FC (Fig. 4 and Table 3). Finally, we validated
two of the computational predicted target genes of
miR-199b-5p, VPS26A and ABCC1, in the tumour set,
that was used for miRNA analysis and demonstrated a
significant increased expression in FC vs NT, PZ0.007
and PZ0.0008 respectively (Fig. 5).
miRNA-based classification of follicular nodules
To provide an overview of the miRNA expressions
across follicular neoplasia (FA and FC) and NT, we
generated a principal component analysis (PCA) using
all expressed miRNAs (Fig. 6A). At this stage, the two
populations could be discerned reflecting the relatively
large and consistent differences in miRNA expression
between the groups. Filtering the expression values by
a t-test, we reached a subset of 179 miRNAs (P!0.01),
where follicular neoplasia could easily be distinguished
from NT tissue. We subsequently attempted to separate
FC from adenoma by the Students t-test for feature
selection and applied the supervised learning
Journal of Molecular Endocrinology (2012) 48, 1123
18 M ROSSING, F N BENNEDBK, F C NIELSEN and others . miRNAs in follicular thyroid tumours

algorithm SVM and LOOCV to generate a classifier. miRNAs, a PCA plot was generated (Fig. 6B). Both the
The optimal signature for classification of FC and FA negative predictive value (NPV) and the positive
consist of two miRNAs, miR-1826 and miR-Eplus-1078, predictive value (PPV) for carcinoma is 83%. The
and based on expression values of the two classification LOOCV was used to compute misclassification rate.

NT FC PC AC
A B
2 (11%)

1 (23%)
3 (9%)

C D

E
2

Figure 2 Heatmaps of predicted miRNA target transcripts in thyroid tumours. (A) depicts
an unfiltered principal component analysis (PCA) of the gene expression in follicular
carcinoma (FC) and normal thyroid tissue (NT). The heatmap in (B) shows the relative
expression of the 260 up-regulated probe sets, representing 135 gene symbols, derived
from the miRNA predicted targets with a composite score OC5 and filtered, so only
differentially expressed genes were included (P!0.01, qZ0.0036) in the analysis. The
heatmap of (C) illustrates the relative expression level of the overlapping 17 transcripts
(GSK3B, FXR1, WAPAL, ITCH, NUPL1, SLC7A11, DDX3X, KPNA4, STK4, MAP3K7IP3,
SSX2IP, TBL1XR1, SIX4, EIF5B, PRPF40A, NARG1 and USP31) represented by 82
probes from the Rodrigues_thyroid_carcinoma_anaplastic_up-regulated_cluster based
on the gene set enrichment analysis. The relative expression of the entire Rodrigues cluster
in FC and NT is depicted in the heatmap of (D). The expression pattern of the 135 miRNA
target transcripts in FC, papillary (PC) and anaplastic carcinoma (AC) is shown in (E).

Journal of Molecular Endocrinology (2012) 48, 1123 www.endocrinology-journals.org

 miRNAs in follicular thyroid tumours . M ROSSING, F N BENNEDBK, F C NIELSEN and others 19

130
Doubling time
for classification of FC and FA consists of 14 miRNAs,
110 Control miRNA 13 h miR-19a, -501-3p, -17, -335, -106b, -15a, -16, -374a, -542-5p,
miR-199b-5p 16 h (P=0004)
90 miR-144 14 h (P=0056)
-503, -320a, -326, -330-5p and let-7i. A PCA plot based
Cell index

70 on expression values of the 14 miRNAs is illustrated

50 in Fig. 6D. Applying this signature resulted in only one
30
misclassified carcinoma and derived a NPV of 92% and
Control miRNA
10
miR-144 a PPV of 100%, both for malignancies.
miR-199b-5p
10
00 210 420 630 840 1050 1260
Hours
Discussion
Figure 3 Effect of proliferation in follicular thyroid cancer cell
transfected with pre-miR-199b and -144. A representative screen- In contrast to papillary and medullary thyroid cancers,
plot from the proliferation-experiment, illustrating a marked
reduction in growth of the follicular thyroid cell line (R082 W-1) where many tumours exhibit defined mutations in
after transfecting with pre-miR-199b. The doubling time of the oncogenes, the causal mutations leading to follicular
transfected cells are 16 h for pre-miR-199b, 14 h, for pre-miR-144
and 13 h for control miRNA. The increase in doubling time is 216036_x_at
significant for the pre-miR-199b transfected cells (PZ0.004) and 203803_at NT
non-significant for the pre-miR-144 (PZ0.056) in comparison with
217576_x_at
235864_at
the doubling time of the control miRNA transfected cells. 217644_s_at FC
222590_s_at
Transfected cells were plated in quadruplicates for each type of 218318_s_at
222589_at
miRNA transfection. The experiment was repeated in biological 212870_at
triplicates. The X-axis represents time in hours and the Y-axis 219439_at
226105_at
represents the normalised cellular index. 218543_s_at
225754_at
224598_at
220189_s_at
Based on 1000 random permutations, the SVM 211613_s_at

classifier comprise a P value of 0.001. The SVM can be

205701_at
211051_s_at
201750_s_at
turned into a probabilistic classifier giving an estimate 219321_at
209109_s_at
of the probability of the predicted class label, i.e. assess 209108_at
225447_at
the prediction uncertainty (Platt 1999). The predictive 223350_x_at
221568_s_at
probabilities for all samples are listed in Table 4. FA 233638_s_at
211665_s_at
sample 11, although correctly classified exhibited a 221618_s_at
219399_at
probability of 0.5 and the misclassified FA sample 12 217301_x_at
214751_at
had a probability for FC of 0.9 indicating that FA_11 is 207606_s_at
210007_s_at
225528_at
highly uncertain, whereas FA_12 is most likely a 224764_at
202444_s_at
misdiagnosed carcinoma. It was not feasible to generate 201807_at
243981_at
a classifier that could distinguish FA and minimally 228483_s_at
225607_at
invasive carcinoma. Even so, widely invasive carcinoma 217262_s_at 2
211085_s_at
can be distinguished from minimally invasive carcinoma 205459_s_at
by the expression of miRPlus-E1001 (P!0.01; average 212714_at
214155_s_at
238960_s_at
threefold up-regulation) and lower expression of miRNA- 1555384_a_at 1
203736_s_at
410 (average fivefold) compared with the minimally 1555355_a_at
57588_at
invasive carcinoma. Lastly, expression values of miR-1826, 202166_s_at
202165_at
miR-Eplus-1078, miR-221, miR-182, miR-96, miR-199b- 203987_at 0
223005_s_at
3p, miR-144* and miR-199b-5p were also examined by 40829_at
203831_at
qRT-PCR and this confirmed the microarray results 238830_at
1558984_at
(Supplementary Figure 1, see section on supplementary 1569791_at 1
1569141_a_at
data given at the end of this article). To validate whether 218030_at
233639_at
miRNAs can classify thyroid follicular malignancies by a 1563657_at
223746_at 2
diverse method, we examined miRNA expression levels
by panels of qRT-PCR assays in 30 (12 FC, 12 FA and Figure 4 Expression patterns of miR-199b-5p target transcripts
in histopathological samples. Two-way cluster illustrating the
6 NT) of the included thyroid samples. An overview
expression of 39 transcripts in follicular thyroid (FC) cancer
based on the total number of expressed miRNAs across all specimens and normal thyroid (NT) tissue. These transcripts are
samples is illustrated in a PCA plot (Fig. 6C). We focused both experimentally verified and computational predicted targets
on building a diagnostic classifier to differentiate between of miR-199b-5p (Fishers exact test, P!0.0005). The expression
FC and FA and found a signature comprising of 14 data are filtered by a P value of 0.05. Blue square represents NT
and FC is labelled in green squares. Each transcript is labelled by
miRNAs to be most favourable. As a consequence of its unique identifier symbol and a list of the equivalent gene
a different miRNA analysing tool, the optimal signature symbols is shown in Table 3.

www.endocrinology-journals.org Journal of Molecular Endocrinology (2012) 48, 1123

20 M ROSSING, F N BENNEDBK, F C NIELSEN and others . miRNAs in follicular thyroid tumours

Table 3 miR-199b-5p target transcripts in follicular cancer specimens. A complete list of 39 target transcripts of miR-199b-5p, matching
the unique identifier probes as illustrated in the two-way cluster, Fig. 4. The listed targets are a subset of the initially 190 computational
predicted targets of miR-199b-5p that were correlated to the down-regulated transcripts in follicular thyroid cancer cells upon transfecting
with pre-miR-199b. This comparison resulted in 56 identical transcripts, i.e. 29.5% of the computational predicted targets were actual
targets. The expression pattern of the 56 transcripts was examined in the data from the histopathological follicular thyroid cancer
specimens and upon filtering the data (P!0.05) the final list resulted in 39 transcripts. Nearly all of the 39 transcripts (87%) were
significantly up-regulated as expected in the follicular thyroid cancer samples

miR-199b-5p target transcripts in follicular thyroid cancer specimens

AP1G1, ARHGAP12, ARHGAP21, CCDC43, CELSR1, C1GALT1, C9orf5, ECE1, ERLIN1, ETS1, EXTL3, FZD6, GIT1, GPD2, IPO8,
LARP4, LIN7C, MAP3K11, MGAT4B, MPP5, NLK, NPAS2, PARP12, PCYOX1, PLXND1, POMGNT1, PPARGC1A, PPFIBP1, PPP1R2,
RBBP4, R3HDM2, SLC24A3, SOS2, STK4, TAF9B, TSPAN6, VPS26A, WDTC1, ZNF468

neoplasia are incompletely understood. Consequently,
efforts have been devoted towards defining biomarkers
such as miRNAs that would allow the clinicians to
distinguish carcinoma from adenoma as well as other
thyroid cancers and obtain information of the molecu-
lar pathways that drive thyroid tumourigenesis.
Compared with NT, FA and FC exhibited widespread
changes in their miRNA expression. We confirmed
previously reported up-regulations of miR-197, -346,
-187, -221, -222, -224 and -155 in carcinoma and
up-regulation of miR-339, -210, -328 and -342 in
adenoma (Weber et al. 2006, Nikiforova et al. 2008)
and identified a number of previously undescribed
thyroid miRNAs including miR-199b-5p, miR-144*,
miR-199b-3p, miR-199a-5p, miR-144, miR-96, miR-182
and miR-597, to mention the most striking. Of
particular significance, among the novel thyroid
miRNAs, miR-199b-5p was found to be lost in the
carcinoma. Loss of miR-199b-5p (also known as miR-
199b) has previously been described in chorioncarci-
noma (Chao et al. 2009) and in meduloblastoma, where
the loss increases metastasis (Garzia et al. 2009). MiR-96
was among the few up-regulated miRNAs in the
carcinoma and this is in line with urothelial carcinomas,
where miR-96 is a promising tumour marker in urine
(Yamada et al. 2010). MiR-182 was also noteworthy in
that increased expression of miR-182 has also been
observed in malignant melanomas and gliomas (Segura
et al. 2009, Jiang et al. 2010), where it is associated with
metastasis and poor prognosis.
However, in order to substantiate the biological
significance of the changed miRNAs, we performed a
weighted target identification (Gallagher et al. 2010),
followed by a molecular pathway analysis and an analysis
of the expression of the target mRNAs, since w80% of
miRNA regulations are reflected by changes in mRNA
expression levels (Guo et al. 2010). Moreover, the
function of miR-199b-5p and miR-144 was examined by
introducing the two miRNAs in thyroid carcinoma cells.
The pathway analyses depicted an enrichment
of transcripts encoding proteins directly involved in
thyroid carcinogenesis and tumourigenesis, and, in the
GSEA, we discovered a significant overlap with genes
up-regulated in AC. The resemblance to AC may not be
surprising, because there is a substantial overlap
between the miRNAs that change in FC and those
altered in AC (Braun et al. 2010). Moreover, in a PCA of
all genes, FCs tend to distribute as a continuum ranging
from relatively well-differentiated FCs to FCs that are
located in close proximity to ACs in contrast to papillary
cancers that form their own homogenous group
(Borup et al. 2010). The common changes in miRNAs
may also be interpreted as an indication that FC and
ACs share a common tumourigenic pathway that differs
from the papillary cancers. Moreover, loss of miRNAs is
likely to represent an early event in follicular neoplasia
since the majority of the miRNAs are also down-
regulated in adenoma. Taken together, the results
imply that the observed changes in miRNA signatures
do have functional consequences in the tumours and
may participate in the tumourigenesis. This was further
reinforced by the demonstration that miR-199b-5p
4
NT
35 FC
3
25
2
15
1
05
0
ABCC1 VSP26A
Figure 5 Relative expression level of two predicted miR-199b-5p
targets by QRT-PCR. Relative expression level of two of the
computational predicted targets of miR-199b-5p, VPS26A and
ABCC1 examined in the tumour set (12 follicular carcinoma (FC)
and 10 normal thyroid (NT) samples) also used for miRNA
analysis. VPS26A and ABCC1 showed a relative increase of
3- (PZ0.007) and 3.5 (PZ0.0008)-fold in FC vs NT respectively.
Blue colour bars represent NT and green ones represent FC.

Journal of Molecular Endocrinology (2012) 48, 1123 www.endocrinology-journals.org

 miRNAs in follicular thyroid tumours . M ROSSING, F N BENNEDBK, F C NIELSEN and others 21

A B
2 (11%)

3 (75%)

1 (98%)

1 (36%)

3 (0%)

2 (2%)

C 2 (9%) D 2 (13%)

1 (33%)

1 (10%)
3 (7%) 3 (12%)

N FA FC

Figure 6 Principal component analysis. (A) shows the projection of follicular carcinomas
(FC) and follicular adenomas (FA) and normal thyroid (NT) employing all miRNAs derived
from the microarray analysis. The projection of FC and FA employing the expression
values of only miR-1826 and miR-Eplus-1078 is seen in (B). (C) shows the projection of
FC and FA and NT employing all miRNAs derived from the qRT-PCR panels. In (D) the
projection of FC and FA employing the expression values of the 14 miRNAs that was
found to be the optimal signature for classification of FC is seen.

reduced cell-growth and that almost 30% of the platform respectively. The qRT-PCR platform provided
computational predicted targets were in fact down- a better separation of FA and FC than the microarray
regulated by pre-miR199b in the cultured thyroid platform, which is reflected by higher accuracy. From a
cells and correspondingly up-regulated in the thyroid clinical point of view, the predicted probabilities
carcinoma lacking the miRNA. MiR-144 had no
significant effect on proliferation and we cannot Table 4 Predictive probabilities of miRNA classifier. Predictive
probabilities for follicular adenoma (FA) and follicular carcinoma
exclude that loss of this miRNA reflects alterations (FC) samples using SVM classifier, Cost Z10, gamma Z0.01.
in the vascularisation of the carcinoma (Rasmussen
et al. 2010). Predictive Predictive
The tumours collected from consecutively referred Sample probability Sample probability
patients whose sex and age were in accordance with that
of larger epidemiological studies. Furthermore, there FC_01 0.04 FA_01 0.73
was no preponderance of oncogenic mutations in the FC_02 0.2 FA_02 0.66
FC_03 0.07 FA_03 0.84
tumour sets. To exploit if the miRNAs could be useful FC_04 0.41 FA_04* 0.45
to depict FC from adenoma, we generated a diagnostic FC_05 0.18 FA_05 0.9
signature from two different technical platforms, the FC_06* 0.67 FA_06 0.94
qRT-PCR assays was included as an independent FC_07 0.13 FA_07 0.93
FC_08* 0.62 FA_08 0.87
validation (Git et al. 2010). Although the results FC_09 0.3 FA_09 0.96
need to be confirmed by independent studies, the FC_10 0.46 FA_10 0.8
performance of either platform was acceptable, as the FC_11 0.22 FA_11 0.5
classifiers exhibited an NPV of 83 and 92% for FC_12 0.08 FA_12* 0.1
malignancies with the microarray and qRT-PCR-based *Misclassified samples (FA: nZ2, FC: nZ2)

www.endocrinology-journals.org Journal of Molecular Endocrinology (2012) 48, 1123

22 M ROSSING, F N BENNEDBK, F C NIELSEN and others . miRNAs in follicular thyroid tumours
derived from each individual sample are essential since
they provide a quantitative value (on the reliability) to
the extent in the accuracy of the diagnosis based on the
classification. According to this, we found that most
samples are classified with high accuracy.
Since we showed that miRNA-based classification
of histopathological follicular thyroid specimens is
possible, the next obvious step is to examine whether
it is feasible to implement miRNA-based classification as
an additional preoperative diagnostic tool. Taking the
limited sensitivity and reproducibility of the histopatho-
logical diagnosis into account, the consistency between
miRNA-based classification and the pathological diag-
nosis is surprisingly high. This could reflect the fact
that all samples were examined by the same dedicated
endocrine pathologist. Studies of inter-observer
variations amongst pathologists in assessment of
follicular lesions have demonstrated an observer
variation for FC of 27%, where the carcinomas tended
to be misdiagnosed as adenomas (Hirokawa et al.
2002, Kakudo et al. 2002). In a similar study an overall
agreement between American and Japanese path-
ologists of 33 and 52% respectively, was found
(Hirokawa et al. 2002).
All results considered, we conclude that thyroid
follicular neoplasia is accompanied by major changes
in miRNA expression that may be directly implicated in
malignant transformation and may facilitate diagnosis
of follicular thyroid cancer.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/
10.1530/JME-11-0039.
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 study was supported by the Agnes and Knut Mrk Foundation,
the Research Foundation of Herlev University Hospital, the Toyota
Foundation and the Novo Nordisk Foundation.
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Received in final form 23 September 2011
Accepted 1 November 2011
Made available online as an Accepted Preprint 2 November 2011
Journal of Molecular Endocrinology (2012) 48, 1123