BioMed CentralMolecular Cancer

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Open AcceResearchEffect of BRAFV600E mutation on transcription and post-transcriptional regulation in a papillary thyroid carcinoma modelSusanne Cahill1, Paul Smyth1, Karen Denning1, Richard Flavin1, Jinghuan Li1, Astrid Potratz2, Simone M Guenther2, Richard Henfrey2, John J O'Leary1 and Orla Sheils*1

Address: 1Dept. of Histopathology, University of Dublin, Trinity College, Dublin, Ireland and 2Applied Biosystems, Foster City, CA, USA

Email: Susanne Cahill - sucahill@tcd.ie; Paul Smyth - smythpa@tcd.ie; Karen Denning - Denningk@tcd.ie; Richard Flavin - flavinr@tcd.ie; Jinghuan Li - jinghual@tcd.ie; Astrid Potratz - astrid.ferlinz@eur.appliedbiosystems.com; Simone M Guenther - simone.m.guenther@eur.appliedbiosystems.com; Richard Henfrey - richard.henfrey@appliedbiosystems.com; John J O'Leary - olearyjj@tcd.ie; Orla Sheils* - osheils@tcd.ie

* Corresponding author

AbstractBackground: microRNAs (miRNAs) are a group of non-coding single stranded RNAs measuringapproximately 22 nucleotides in length that have been found to control cell growth, differentiationand apoptosis. They negatively regulate target genes and have recently been implicated intumourigenesis. Furthermore, miRNA expression profiling correlates with various cancers, withthese genes thought to act as both tumour suppressors and oncogenes. Recently, a point mutationin the BRAF gene leading to a V600E substitution has been identified as the most common geneticchange in papillary thyroid carcinoma (PTC) occurring in 29–69% of cases. This mutation leads toaberrant MAPK activation that is implicated in tumourigenesis.

Aim: The aim of this study was to identify the effect that BRAF oncogene has on post-transcriptional regulation in PTC by using microRNA analysis.

Results: A unique miRNA expression signature differentiated between PTC cell lines with BRAFmutations and a normal thyroid cell line. 15 miRNAs were found to be upregulated and 23 miRNAswere downregulated. Several of these up/down regulated miRNAs may be involved in PTCpathogenesis. miRNA profiling will assist in the elucidation of disease pathogenesis andidentification biomarkers and targets.

BackgroundPapillary thyroid carcinoma (PTC) is the most commonlyoccurring thyroid malignancy accounting for approxi-mately 80% of cases. Genetically PTC is defined by severalalterations which cause abnormal activation of themitogen-activated protein kinase (MAPK) pathway, the

most prevalent being point mutations in the intracellularsignalling kinase BRAF [1,2]. An activating mutation inBRAF (V600E) leads to constitutive activation and hencede-regulation of MAPK pathway. Several groups havereported that PTCs harbouring mutations in BRAF havemore aggressive properties, present more often with extra-

Published: 13 March 2007

Molecular Cancer 2007, 6:21 doi:10.1186/1476-4598-6-21

Received: 13 December 2006Accepted: 13 March 2007

This article is available from: http://www.molecular-cancer.com/content/6/1/21

© 2007 Cahill et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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thyroidal invasion and also at a more advanced stage. His-tological features such as the classic PTC architecture andtall cell features are also associated with the BRAF onco-gene [3,4].

Despite this range of knowledge thyroid carcinoma stillposes a significant diagnostic challenge and understand-ing of the complete range of genes and pathways involvedin its pathogenesis is incomplete. Until recently biomar-ker discovery has concentrated largely on protein codingRNAs in a quest to identify cancer associated genesinvolved in thyroid carcinogenesis [5-7]. The recent dis-covery however that a large group of non-coding RNAscalled microRNAs (miRNAs) may potentially play a rolein cancer has led to a proliferation of miRNA relatedresearch of late including the publication of several keyreviews [8-12]. These small non-coding RNAs constitute anovel class of gene regulators that function by negativelyregulating gene expression by targeting mRNAs for cleav-age or translational repression. Several recent studies onthe regulatory functions of miRNAs suggest that they playcritical roles in central processes such as development, cellproliferation and differentiation, stress resistance, metab-olism and apoptosis all of which are involved in tumour-igenesis [13,15]. Not surprisingly aberrant miRNAexpression has been implicated in cancer developmentand a hypothesis has emerged suggesting that tumoursmay each have a discrete "miRNA signature" [16-20]. Dif-ferential expression of miRNAs between malignant tissueand normal tissue and between different types of tumour,has been shown in several key studies, indicating thatmiRNAs are determinants of clinical diagnostic and prog-nostic significance [21,22].

Recently several groups have utilised a range of techniquesmost notably northern blot analysis, cloning and oligonu-cleotide microarrays, in order to analyse miRNA expres-sion profiles in different cancer types [23-26]. In thisstudy a novel stem-looped TaqMan® RT-PCR method wasused to measure the expression levels of a panel of 160mature miRNAs in thyroid cell lines containing BRAFmutation.

ResultsDifferential miRNA expression using TaqMan™ microRNA assaysStem-looped TaqMan® RT-PCR was used to measure theexpression levels of 160 miRNAs in a collection of celllines including a normal thyroid cell lines N-thy-ori 3-1and two cell lines containing V600E BRAF point mutation[Nthy-oriBRAF and KAT-10]. A comparison was thenmade between Nthy-ori and BRAF mutated cell lines todetermine miRNAs whose expression was differentiallyexpressed between the two groups. ∆∆CT method was usedto calculate fold change of miRNA expression between

groups. 15 miRNAs were found to be upregulated with afold change of > 2 in BRAF mutated cell lines when com-pared to normal.

Three of these upregulated miRNAs showed significantlyhigher fold change than the other upregulated miRNAs,these are mir-200a, mir-200b and mir-141.

23 miRNAs showed underexpression of > 2 fold whencompared to the normal thyroid cell line. Moreover, threemiRNAs showed significantly more underexpressioncompared to the other downregulated miRNAs. ThesemiRNAs are as follows mir-127, mir-130a and mir-144.(Table 1 and Figures 1 and 2).

mRNA (transcription) and miRNA (regulation) expression correlationmiRNAs interact and regulate their target genes by cleav-age or translational inhibition [12]. A current challenge isto identify biologically relevant targets that are regulatedby individual miRNAs. This task is further complicated bythe fact that each miRNA can potentially bind and regu-late many mRNA targets and each mRNA can be boundand regulated by several miRNAs. Several publicly availa-ble databases exist for prediction of miRNA targets. MiR-Base, PICTAR and TARGETSCAN were used in this study[26-28].

These three databases were utilized to yield lists of puta-tive targets for each miRNA which are illustrated in table

Table 1: Differentially expressed miRNAs in BRAF mutated cell lines V normal

Upregulated Downregulated

has-mir-128a hsa-mir-122ahas-mir-128b hsa-mir-127has-mir-135a hsa-mir-130ahas-mir-141 hsa-mir-137has-mir-150 hsa-mir-138has-mir-185 hsa-mir-144has-mir-200a hsa-mir-155has-mir-200b hsa-mir-181bhas-mir-200c hsa-mir-187has-mir-203 hsa-mir-190has-mir-213 hsa-mir-193has-mir-215 hsa-mir-197has-mir-330 hsa-mir-222has-mir-338 hsa-mir-302bhas-mir-34a hsa-mir-302c

hsa-mir-323hsa-mir-335hsa-mir-339hsa-mir-342hsa-mir-34chsa-mir-370

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2. From these lists there are several genes that are biologi-cally relevant in the context of PTC. The miRNAs whichshowed the highest increase in fold change are predictedto bind genes involved in thyroid function, MAPKKK cas-cade, retinoic acid receptors, cell adhesion and cell struc-ture associated genes and genes involved in cell signalling.miRNAs which showed the largest decrease in fold changeare predicted to regulate and target genes involved in G-protein mediated signalling, oncogenesis and cell cyclecontrol. One might predict that a miRNA which is overex-pressed would be associated with downstream underex-pression of its target. This regulation can occur either atthe transcript level or the protein level.

DiscussionmicroRNAs (miRNAs) are now recognized as an addi-tional layer of post-transcriptional control that must beconsidered in order to understand the complexity of geneexpression in humans. miRNAs are now known as a novelclass of gene regulators that are involved in cancer related

processes and deregulation or aberrant expression of miR-NAs may contribute to human diseases, including cancer.Calin et al showed down-regulation of miRNA-15 andmiRNA-16 in a majority of chronic lymphatic leukaemia(CLL). Altered miRNA expression has also been reportedin breast cancer, glioblastoma, lung cancer and colorectalcancer [29-35].

This study utilized a novel stem-looped TaqMan RT-PCRmethod to measure the expression levels of 160 maturemiRNAs in a collection of thyroid cell lines. This methodoffers several distinct advantages over conventionalmiRNA detection methods including a higher sensitivityand specificity and a fast and simple methodology. Theaim of this study was to correlate BRAF mutant status withan associated miRNA profile, with a view to elaboratingthe downstream regulatory effect of BRAF mutation.

The data showed a set of differentially expressed miRNAsin cell lines with BRAF V600E mutation compared to a

Differentially expressed miRNAs Between BRAF V600E harbouring Cell Lines and Normal Thyroid Cell lineFigure 1Differentially expressed miRNAs Between BRAF V600E harbouring Cell Lines and Normal Thyroid Cell line. Delta delta CT was performed using Nthy-ori 3-1 as a normal control. The log of the RQ values was used to plot the relative fold change of Nthy-BRAF and KAT10 against Nthy-ori 3-1. miRNAs are on the × axis

-4

-3

-2

-1

0

1

2

3

4

LOGRQ

hsa-mir-122a

hsa-mir-127

hsa-mir-128a

hsa-mir-128b

hsa-mir-130a

hsa-mir-135a

hsa-mir-137

hsa-mir-138

hsa-mir-141

hsa-mir-144

hsa-mir-150

hsa-mir-155

hsa-mir-181b

hsa-mir-185

hsa-mir-187

hsa-mir-190

hsa-mir-193

hsa-mir-197

hsa-mir-200a

hsa-mir-200b

hsa-mir-200c

hsa-mir-203

hsa-mir-205

hsa-mir-210

hsa-mir-213

hsa-mir-215

hsa-mir-222

hsa-mir-302b

hsa-mir-302c*

hsa-mir-323

hsa-mir-330

hsa-mir-335

hsa-mir-338

hsa-mir-339

hsa-mir-342

hsa-mir-34a

hsa-mir-34c

hsa-mir-370

NTHYNTHYBRAFKAT10

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Heat map of MiRNA expressionFigure 2Heat map of MiRNA expression. miRNA expression. Each miRNA listed was detected as significantly differentially expressed between BRAF mutated cell lines and N-thy-ori. The delta CT values for each miRNA were used to create the heat map

hsa-mir-122ahsa-mir-127hsa-mir-128ahsa-mir-128bhsa-mir-130ahsa-mir-135ahsa-mir-137hsa-mir-138hsa-mir-141hsa-mir-144hsa-mir-150hsa-mir-155hsa-mir-181bhsa-mir-185hsa-mir-187hsa-mir-190hsa-mir-193hsa-mir-197hsa-mir-200ahsa-mir-200bhsa-mir-200chsa-mir-203hsa-mir-205hsa-mir-210hsa-mir-213hsa-mir-215hsa-mir-222hsa-mir-302bhsa-mir-302c*hsa-mir-323hsa-mir-330hsa-mir-335hsa-mir-338hsa-mir-339hsa-mir-342hsa-mir-34ahsa-mir-34chsa-mir-370

Molecular Cancer 2007, 6:21 http://www.molecular-cancer.com/content/6/1/21

normal thyroid cell line (Table 1). There were 15 overex-pressed miRNAs and 23 underexpressed miRNAs in thesecell lines compared to the normal cell line. Of these miR-NAs listed in table 1 only a few have been previously asso-ciated with other cancers. One of which is miR-127, thatshowed down-regulation in BRAF V600E mutated celllines. Human miR-127 is embedded in a CpG island andis silenced in several human cancers including bladder,prostate and colon cancer and has been suggested to playa role as a possible tumour suppressor gene [33]. miR-127also an example of the potential utility of miRNA targetedtherapy in cancer. Saito et al demonstrated that inductionof miR-127 by with 5-aza-20-deoxycytidine and 4-phenyl-butyric acid, which inhibits DNA methylation and his-tone deacetylase respectively, reduces expression of theoncogene BCL6 in bladder cancers [33]. Its utility as apotential cancer target has yet to be explored in other can-cers however this suggests the possibility of direct target-ing of miRNAs that are amplified or upregulated inpatient tumours. Two other miRNAs listed in table 2 havealso been described in other cancers. These include miR-200b and miR-141 which have been shown to be highlyoverexpressed in malignant cholangiocytes and in coloncarcinoma. [18]. This suggests the possibility of miR-200band miR-141 down-regulating possible tumour suppres-sor genes. Of particular interest in this study is the down-regulation of miR-323 and miR-302b in BRAF mutatedcell line. miRBASE target database predicted that the BRAFtranscript has binding sites for miR-323 and miR-302b topotentially bind and down- regulate BRAF expression.

A number of miRNAs exhibited a difference in expressionlevel between KAT10 cell line and normal but did notshow a significant change in the BRAF transfected celllines and normal. miRNAs specific to the PTC cell linewith native mutant BRAF included, mir-10a [overex-pressed with a fold change of 95 in KAT10] mir-23b[downregulated with a fold change of 2949] and mir-154[downregulated with a fold change of 142]. It is possiblethese miRNAs may play a role in a later stage in tumourformation and progression; given the transfected cell line

had only been exposed to mutant BRAF for a short period(3 passages) unlike KAT10 which represents a genuinePTC which has evolved with BRAT mutant as an intrinsiccomponent. Alternatively, the difference may reflect theaccumulation of several genetic insults in the process oftumourigenesis.

Recent studies in miRNA deregulation PTC found an aber-rant miRNA expression profile in PTCs compared to nor-mal thyroid tissues [36,37]. In particular, a significantincrease in mir-222, mir-221, mir-146. Our study did notfind any of these miRNAs to be significantly overex-pressed in the cell lines examined. miR-222 was actuallyfound to be downregulated in the BRAF mutated celllines. Another miRNA these two groups found to beupregulated albeit less significantly was miR-213, ourstudy also found miR-213 to be upregulated in BRAFmutated cell lines [25 fold]. These differences may be dueto the different experimental approaches; Huiling et aland Pallante et al used miRNA microarrays. They alsoused different chemistry and fresh tissue samples; also theRET and BRAF status of each PTC case was not disclosed.

Prediction of miRNA targets is an immediate challenge inmiRNA research. Several computational approaches havebeen implemented for miRNA gene prediction usingmethods based on sequence conservation and/or struc-tural similarity. To overcome the limitations of target pre-diction three of these databases MiRBase, TARGETSCANand PICTAR were used in this study to predict possible tar-gets of the differentially expressed miRNAs associatedwith BRAF mutated (Table 2). Examples of other pro-grams developed for target identification include RNAhy-brid, RNA calibrate and RNAeffective. Only a few targetmRNAs have been experimentally proven and studied invitro and not all the prediction algorithms yield overlap-ping lists therefore the lists may contain several false pos-itives that may fit the criteria but do not interact with themiRNA in vitro. To overcome this, results were intersectedfrom these databases to identify the genes commonly pre-dicted by at least two of the algorithms. The results of the

Table 2: Predicted targets for differentially expressed miRNAs

miR-200a IRS2, MAP3KA, MYH10, SLC20A1, YWHAG, E2F3, RAB38, PPP2R2A, CALCR, MTSS1, RAB30, STXBP1, THRAP2, STRN, MATR3, MTPN, FGFR2

miR-128b PLK2, PRKD1, ADORA2B, ZNF385, CASC3, PAIP2, ITGB4BP CRKL, CDH24, ING5, BAG2, EGFR, ST14, THRAP1 CORO1CE2F, E2F3, PDGFRA, INSR, CITED2, STX16

miR-141 PDCD4, MYH10, WTAP, STAT5A, PERP, TCF8, FUS, MYL4 LBR, SOCS6, ACTA,1MLANA, CDK2, MAGED4, EIF3S9, HISTIH2AG KRT10, XPO4, DCTN3

miR-127 RIMS4, TLK2, BCL6, TFF1, VASH1, PCDH21, BAD, TTGB5, ICAM 3, TMEMG, HIST4H4, APOB, LGALS8, CCNK,miR-34c RAB43, CELSR3, NOTCH1, MET, CALCR, NOTCH2, THRAP2, TPO52, DAAMI, PDGFRA, MAD1AmiR-302c RAB43, CELSR3, NOTCH1, MET, CALCR, NOTCH2, THRAP2, TPO52, DAAMI, PDGFRA, MAD1A

miR-200a, miR-128b, miR-141 = Upregulated miRNAmiR-127, miR-34c, miR-302c = Downregulated miRNA

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potential targets of several differentially expressed miR-NAs are displayed in table 2.

It is reasonable to expect that targets of downregulatedmiRNAs include oncogenes or genes encoding proteinswith potential oncogenic function. Indeed among puta-tive targets, several genes with potential oncogenic func-tions were found include RAB35, ERBB4, MAP3K3,VASP6, KIT, FOSB, NOTCH1. Upregulated miRNAs arepurported to interact with tumour suppressor genes.Those identified from this study include MAD 4 and ST6.Several extracellular membrane molecules were also iden-tified including; myotubulin, MAD1A, Cadherins. In gen-eral overexpression of an miRNA would be associatedwith downregulation of its target miRNA.

Interestingly among these potential gene targets are anumber of genes that have implications in thyroid carci-noma progression such as genes involved in thyroid func-tion, cell cycle control, cell signalling, MAPKKK cascade,retinoic acid receptors, cell adhesion and cell structureassociated genes. Therefore, it seems plausible that thissubset of miRNAs may be important in the progression ofthyroid carcinoma by working together or independentlyto regulate genes involved in thyroid tumourigenesis

Our group has also previously examined gene expressionlevels in these same cell lines. This analysis was done by

evaluating genome-wide gene expression levels in BRAFmutated cell lines and matched normal thyroid cell linesusing the Applied Biosystems Gene Expression Arrays.Treatment comparisons were preformed resulting in listsof differentially expressed genes (Figure 3). These genelists were uploaded into PANTHER (Protein ANalysisTHrough Evolutionary Relationships) classification sys-tem and the software statistically compared them to a ref-erence list to look for under and over-representedmolecular functions and biological process. Analysisrevealed a set of genes associated with mutant BRAFexpression which are involved in processes such as apop-tosis, chromosomal instability, invasiveness and metasta-sis and proliferation. Other genes identified were shownto be involved in the insulin-like growth factor pathway(including (insulin-like growth factor binding protein)IGFBP4, (insulin receptor substrate) IRSI, GRB14, PLAG1and IGF2) and the Wnt signaling pathway suggesting theirimportance in BRAF signalling.

Up and down regulation of cell adhesion and chromo-some remodelling molecules were also shown. BRAFmutation has previously been shown to induce chromo-some instability and decrease cell- cell interactions [38]and our data further corroborates the involvement ofthese processes in papillary thyroid carcinoma. Not sur-prisingly genes involved in the MAPK pathway, of whichBRAF is an integral part, showed an increase in expression.

Hierarchical Clustering of BRAF mutated Cell linesFigure 3Hierarchical Clustering of BRAF mutated Cell lines. In (a) The column dendrogram clearly shows cases clustering on the basis of BRAF mutation. To the left are the normal thyroid cell lines with no BRAF mutation and to the right are Nthy-BRAF with V600E mutation. In (b) The denodrogran shows cases again clustering on the basis of BRAF mutation. To the left are the PTCs with BRAF V600E mutation and to the right are the normal thyroid cell lines with no mutation.

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Among these genes were DDIT, DUSP6, TFPI-2, Seladin-1,MAP3K12, and GRB14. These results provided an insightinto the different pathways through which BRAF exerts itsoncogenic effect and emphasised the importance of theMAPK and Wnt signalling pathways in thyroid tumourformation.

The list of putative miRNA targets (as defined above intable table 1) correlated with the list of differentiallyexpressed genes on analysis. This is illustrated in table 3.It is interesting to speculate that these genes are regulatedby their corresponding miRNA thereby contributing totumour development in thyroid. These observationsshould be placed in context, in so far as predictive data-bases are incomplete and target selection is essentiallyspeculative and involves a degree of subjectivity as thesetarget mRNAs have not been experimentally proven andstudied in vitro.

In summary this study revealed a set of miRNAs associatedwith expression of mutant BRAFV600E oncogene in PTC celllines when compared to a normal thyroid cell line. TheBRAF oncogene may influence miRNA expression directlyor indirectly by altering the expression patterns of genes,such as transcription factors, involved in tumour progres-sion or indeed the genes involved in the biogenesis andsynthesis of miRNAs such as those from the RISC complex

and miRNA machinery. These miRNAs associated withBRAF oncogene as listed in Table 1 may contribute totumour formation by regulating genes involved in theMAPK pathway in which BRAF plays a role.

This data suggests the potential utility of miRNA expres-sion patterns as a molecular screen for PTC to use as adiagnostic adjuvant to already established morphologicalcriteria. A recent study by this group examined the effectthe ret/PTC 1 oncogene has on miRNA expression in PTCcell lines. ret/PTC 1 rearrangements are generally associ-ated with classic papillary architecture and indolentbehaviour in thyroid carcinoma. This study showed a dis-tinct pattern of miRNAs associated with the ret/PTC 1oncogene when compared to a normal thyroid cell lines[39].

It is noteworthy that different miRNA expression profileshave been shown contingent upon ret/PTC 1 activatedand BRAF mutated cell lines. This suggests that miRNAexpression analysis may also be of prognostic significance.Further miRNA analysis in a larger cohort of samples willhelp validate and consolidate the results discussed above.Table 4 illustrates a compilation of miRNAs that are differ-entially expressed in PTC cell lines (either ret/PTC-1 orBRAF mutant), suggesting an important role for thesemiRNAs in thyroid neoplasia progression independent of

Table 3: miRNAs and Predicted Targets.

Up-regulated Genes Potential Binding Partner (down-regulated)

PGM5 miR-137DUSP6 miR-138TLE miR-323HSPH1 miR-181bPCGF1 miR-138CDK2AP2 miR-302c, miR-302bDDIT3 miR-144SLC14A1 miR-222MANEA miR-302b, miR-302cELP4 miR-122aAPIP miR-181bPEX7 miR-190

Down-regulated Genes Potential Binding Partner (up-regulated)

SYTL2 miR-128a, miR-128bGJB3 miR-34aSDC4 miR-141, miR-215CREBL2 miR-141, miR-200a,CCL2 miR-141, miR-200aGYG2 miR-215HIATL1 miR-34aZMYND11 miR-200aADAMTS4 miR-141

Differentially expressed miRNAs in BRAF mutated cell lines. Red = Upregulated. Green = Downregulated

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molecular trigger. They may be involved in regulating thecentral MAPK pathway involved in thyroid tumourigene-sis in which RET and BRAF oncogenes play a part.

ConclusionAlthough the exact function of many miRNAs remains tobe elucidated it is clear that miRNAs potentially have abroad influence over several diverse genetic pathways andthat their deregulation is likely to contribute to diseaseincluding cancer. An exciting future prospect is that themiRNA patterns associated with a particular tumour mayultimately be of diagnostic and prognostic significanceand contribute to the understanding of the molecularpathogenesis and gene regulatory processes of cancer.

Materials and methodsCell culture and transfectionNthy-ori 3-1 (ECACC, Wiltshire, UK) is a thyroid follicu-lar epithelial cell line derived from normal thyroid tissueof an adult that has been transfected with a plasmidencoding for the SV40 large T gene. KAT10 is derived froma papillary thyroid carcinoma cell line with heterozygousBRAF V600E mutation. Both cell lines were grown to con-fluence in a humidified atmosphere containing 5% CO2 at37°C in the following plating medium: RPMI 1640 with2 mM L-glutamine, 10% Foetal calf serum (FCS), Penicil-lin (100 U/ml) and Streptomycin (100 µg/ml). A BRAFV600E expressing plasmid, pMCEF-V600EB-RAF, wastransfected into Nthy-ori 3-1. Transfections were carriedout using Genejuice™ transfection agent (Novagen, Ger-many) using the recommended protocol and transfectedcells were grown in the presence of Gentamicin (SigmaAldrich) antibiotic to yield pure cultures.

Nucleic acid extractionFollowing trypsinisation of cultured cells, cell pellets (3–4 × 106 cells) were collected and total RNA was extractedusing RNeasy® mini kit (Qiagen Ltd., West Sussex, UK).RNA quantity and quality are assessed using a NanoDrop®

ND-1000 Spectrophotometer (Wilmington, USA) andRNA 6000 Nano LabChip® Kit in conjunction with theAgilent 2100 Bioanalyser (Agilent technologies, Wald-bronn, Germany) as illustrated in figure 4. Taqman® SNPdetection was used for BRAF V600E mutation detection as

previously described by Smyth et al [40]. Primers andprobes used in this experiment were designed and used tothe manufacturer's recommendations. The primers/probes used were as follows: 5' CAT GAA GAC CTC ACAGTA AAA ATA GGT GAT 3' [BRAF-F], 5' GGA TCC AGACAA CTG TTC AAA CTG A 3' [BRAF-R], VIC-5' CCA TCGAGA TTT CAC TGT AG 3' [BRAF-PWT], and FAM-5' CCATCG AGA TTT CTC TGT AG 3' [BRAF-PMUT]. Amplificationand analysis was performed using an ABI Prism 7000Sequence Detection System (Applied Biosystems, CA,USA) for 40 cycles (92°C for 15 sec, 60°C for 1 min).

Western blot analysis was carried out on the transfectedcell lines to confirm BRAF protein expression.

Bioanalyser "virtual" gel electrophoresis for N-thy-ori Cell LinesFigure 4Bioanalyser "virtual" gel electrophoresis for N-thy-ori Cell Lines. Lane 1: Nthy-ori rep1; Lane 2:Nthy-ori rep 2; Lane 3: Nthyori rep3

Table 4: Differentially Expressed miRNA in ret/PTC 1 and BRAF mutated cell lines

Upregulated miRNAs Downregulated miRNAs

miR-128a miR-127miR-128b miR-302bmiR-185 miR-302cmiR-200a miR-323miR-200b miR-370miR-213

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Microarray analysisThe Applied Biosystems 1700 Expression Array System isbased on a microarray design that represents the wholehuman genome. The V.2 array has 32,878 60-mer oligo-nucleotide probes for the interrogation of 29,098 individ-ual human gene and more than 1,000 control probes. V2arrays were used to analyse the transcriptional profiles ofthe cell line RNA samples in this study. Digoxigenin-UTPlabeled cRNA was generated and linearly amplified from2 µg of total RNA using Applied Biosystems Chemilumi-nescent RT-IVT Labelling Kit v 2.0 following the manufac-turer's protocol. Array hybridization, chemiluminescencedetection, image acquisition and analysis were performedusing Applied Biosystems Chemiluminescence DetectionKit and 1700 Chemiluminescent Mircoarray Analyzer fol-lowing manufacturer's guidelines. Each microarray wasinitially pre-hybridised at 55°C for 1 hr in hybridizationbuffer with blocking reagent. 10 µg of labeled cRNA tar-gets were fragemented by incubating with fragmentationbuffer at 60°C for 30 min, mixed with internal control tar-get (ICT, 24-mer oligo labeled with LIZ fluorescent dye)and hybridized to each pre-hybed microarray in a 1.5 mlvolume at 55°C for 16 hr. After hybridization, the arrayswere washed with hybridization wash buffer and chemi-luminscence rinse buffer. Enhanced chemiluminescenctsignals were generated by incubating arrays with anti-dig-oxigenin alkaline phosphatase, enhanced with Chemilu-minescence Enhancing Solution and finally by addingChemiluminescence Substrate. Images were collected foreach microarray using the 1700 analyser. Images wereautogridded and the chemiluminescent signals werequantified, corrected for background and spot and spa-tially normalized. Replicates were performed. Microarrayswere analysed using Spotfire Decision Site™ for functionalgenomics (Spotfire AB, Göteborg, Sweden) and R version1.9.1 [a free language and environment for statistical anal-ysis and graphics] (R Development Core Team, 2004).

miRNA analysisApplied Biosystems TaqMan® microRNA (miRNA) assaysare designed to detect and quantify mature miRNAs usinga looped-primer real time PCR. The human early accesspanel used in this study contained 160 individual assaysfor identified human miRNAs. The assay involved 2 steps:Step one; a Stem-looped RT, and Step two; a Real Time

PCR. Briefly, single stranded cDNA was generated fromtotal RNA sample by reverse transcription using theApplied Biosystems High-Capacity cDNA Archive Kit(Applied Biosystems, CA, USA) following manufacturer'sprotocol. RT reactions contained 10 ng of total RNA, 50nM stem-looped RT primer, 1 × RT buffer, 0.25 mM each

of dNTPs, 3.33 U/µl Multiscribe reverse transcriptase and0.25 U/µl RNase Inhibitor. PCR amplification was carriedout using sequence specific primers on the Applied Bio-systems 7900 HT Fast Real-Time PCR system. The reac-tions were incubated in a 96-well optical plate at 95°C for10 min, following by 40 cycles of 95°C for 15 s and 60°Cfor 10 min. Analysis of relative miRNA expression datawas performed using ∆∆CT method with hsa-let-7a as anendogenous control [41]. Two negative controls were alsoused, ath-mir159a and cel-lin-4 as they show no expres-sion in human tissue.

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