its morphological features resemble undifferentiated neoplasm . due to severe metastasis development and to the rapid fatal course
radiotherapy and chemotherapy are also not very effective . it has been suggested that those standard therapies are ineffective because they are not able to efficiently target a subpopulation of atc cells , called the cancer - initiating cells or cancer stem cells ( cscs ) . it has been proposed that cscs possess stem - cell - like features , are at the core of the development of many tumours , especially undifferentiated ones like atc , are responsible for the recurrence of the tumour and metastasis formation , and usually are very resistant to classical therapies . despite many controversies regarding the cancer stem cell model , it has the potential to drive the discovery of innovative treatments that may eradicate the very chemoresistant core of cancer . in this connection ,
the csc model is the sum of many hypotheses that have arisen to explain the most vexing aspects of cancer : metastasis , relapse , and therapeutic resistance . in this perspective
, csc research holds out promise for improved treatment outcomes , in particular , as regards overcoming resistance to chemotherapy on solid tumours .
the most accepted csc model makes use of a new paradigm of cellular differentiation , in which cancer cells can dedifferentiate toward more primitive , stem - like phenotypes .
the dedifferentiation seems to be highly heterogeneous , giving an explanation to the observed discontinuous behaviour of many cancers .
alternatively , cscs might arise from transformed stem cells in the stem niche [ 1 , 2 ] . similar to normal stem cells , cscs have the ability both to self - renew and to give rise to differentiated tumour cells , are responsible for the organization of a tumour mass , and are tumorigenic when transplanted into an animal host .
cscs have been identified in a wide range of human tumours . at the molecular level , cscs are usually enriched in cell surface markers such as cd44 , cd24 , and cd133 , while wnt/-catenin , notch , and hedgehog signalling pathways seem to have key roles in csc properties .
specific microrna signatures have been identified in many cscs   that seem to play a role in the epithelial - mesenchymal transition . regarding atc , it has been hypothesized that the tumour initiates from transformed thyroid stem cells , rather than from differentiated thyrocytes undergoing a conventional multistep carcinogenesis model [ 57 ] .
the rarity and rapid fatal nature of atc has led to limited ex vivo studies .
here we describe an in vitro study on a well - validated atc cell line : sw1736 .
the sw1736 cell line is characterized by a high percentage of population with stem cell - like properties and high expression of several stem markers ( sox2 , oct4 , nanog , c - myc , ssea4 , and the abcg2 transporter ) .
interestingly , sox2 silencing downregulates in trans the expression of other stem cell markers and sensitizes atc cells to treatment with classical chemotherapeutics such as cisplatin and doxorubicin .
this suggests that the stem cell factor sox2 could have a preeminent hierarchical role in determining the characteristics of stem cells in sw1736 atc cell line .
therefore , in order to further investigate the role of sox2 in atc , a bioinformatic analysis of the functional network of sox2 was performed . in detail ,
this kind of analysis is able to predict genes functionally correlated with the bait gene rather than physically associated with it [ 9 , 10 ] .
the cerna hypothesis is based on the rationale that rna molecules can regulate one another via micrornas [ 9 , 10 ] .
cernas are rnas that share mirna recognition elements , thereby regulating each other by influencing the available level of mirna [ 9 , 10 ] . in the past ,
cerna analysis made it possible to isolate several genes and functional networks related to cancer development , ageing , and homeostasis [ 1119 ] .
mirwalk is a comprehensive database that provides information on mirna from humans , mice , and rats on their predicted as well as validated binding sites on their target genes .
using the mirwalk   data and embedded tools , we collected the micrornas that have been reported in the literature to regulate the main transcript from the sox2 locus ( table 1 ) .
this set of mirnas was inserted into the mirwalk analysis tool   to collect any human mrna that has been reported to be regulated by them .
then the genes collected were organized in a hierarchical order for the number of validated microrna hits ( table 2 ) .
the more micrornas are shared between the bait sox2 gene and the candidate genes , the higher the possibility that the candidate gene transcripts can act as sox2 cernas .
arbitrarily , the top 6 genes together with sox2 were analysed using the genemania   tool that helps to predict the functions of a set of genes and to predict in which gene ontology ( go ) functions the set of genes might be involved ( figure 1 ) ( table 3 ) .
the sw1736 , 8505c , c643 , fro , bcpap , tpc-1 , and wro cell lines were cultured in dulbecco 's modified eagle 's medium high glucose medium supplemented with 10% fetal bovine serum and 5% glutamine .
sirna transfection in sw1736 cells was performed using interferin transfection agent ( polyplus - transfection , illkirch , france ) , according to the manufacturer 's instructions .
briefly , the transfection agent and the sirna complex were added to the cells and incubated for 72 hours for rna extraction and analysis .
sox2 was silenced using stealth sirna sox2 hss144045 ( invitrogen , milan , italy ) .
sicontrol stealth sirna negative control was used as a control ( invitrogen , milan ) .
the vector used was taken from addgen ( http://www.addgene.org/ ) ( plasmid 26817 ) : pcdna3.3_sox2 ; and transformation into sw1736 cells was performed using xfect transfection agent ( clontech laboratories , inc . a takara bio company ) according to the manufacturer 's instructions .
the transfection agent and plasmid were added to the cells and incubated for 72 hours for rna extraction and analysis .
the vector was synthesized in service by eurofins genomics ( https://www.eurofinsgenomics.eu ) using a pcdna 3.1 backbone and a chemically synthesized 3utr ( as reported in http://mybioinfo.info/exon_display.php?tax_id=9606&gene_id=geneid : 6657 ) ( table 4 ) .
total rna was extracted from cells using the rneasy mini kit ( qiagen , milan , italy ) , including a digestion step with dnase i. rna quantity and quality were assessed using the nanodrop 2000 ( thermo scientific , wilmington , usa ) .
the rna extracted was reverse - transcribed with random hexamers ( applied biosystems , darmstadt , germany ) and improm ii reverse transcriptase ( promega italia , milan , italy ) , according to the manufacturer 's protocol .
the reactions were performed as follows : 5 at 94c , 30 cycles ( 30 at 94c , 30 at 55c , 30 at 72c ) , 5 at 72c , and stocked at 4c .
the only exception was the amplification of the sox2 3utr , for which the following was done : 5 at 94c , 30 cycles ( 30 at 94c , 30 at 55c , 90 at 72c ) , 5 at 72c , and stocked at 4c .
expression was analyzed by real - time quantitative pcr ( qrt - pcr ) using quantitect sybr green pcr kit ( qiagen , milan , italy ) .
all reactions were performed using a rotor - gene q instrument ( qiagen , milan , italy ) .
a pool of rna from normal thyroid tissue specimens was used , as described in .
a pool of rna from limbal stem cells specimens was used , as described in .
peripheral blood samples of a healthy volunteer were collected in tubes containing ethylenediaminetetraacetic acid ( edta , 1  mg / ml ) after 8 hours ' fasting . lymphocytes were isolated by lympholyte ( cedarlane , burlington , ontario , canada ) , according to the manufacturer 's instructions .
we used the spss 13 software , windows edition , for all our statistical analyses .
correlations were determined using spearman 's test ( nonparametric equivalent for pearson 's test ) .
previous data   suggested that the stem cell factor sox2 possesses a preeminent hierarchical role in determining stemness characteristics in the sw1736 atc cell line . with the final aim of investigating the role of sox2 in atc , a bioinformatic cerna analysis [ 9 , 10 ] of the functional network of sox2
, we collected the micrornas that have been reported in the literature to regulate the main transcript from the sox2 locus ( table 1 ) .
this set of mirnas was inserted into the mirwalk analysis tool   to collect any human mrna that has been reported to be regulated by them . then the collected genes were organized in a hierarchical order for the number of validated microrna hits ( table 2 ) .
the more micrornas are shared between sox2 and the candidate genes , the stronger the putative competitive effect that is at the core of the cerna hypothesis .
the top level sox2 interactors in this cerna analysis are dicer1 , eif2c2 , and rnasen , involved in mirna biogenesis and functions ; the most studied antioncogene tp53 , worthy of note because of its suggested role in stemness ; the nuclear - coded mitochondrial cytochrome c oxidase subunit viiia cox8a ; and ccnd1 , the cyclin d coding gene . amongst the lesser interactors reported in table 2
, other genes might be worth studying in the future , especially for their involvement in oncogenesis ( such as myc , bcl2 , pten , kras , jun , and many others ) .
this study aimed to analyse whether a relationship might exist between the 6 top level interactors ( dicer1 , eif2c2 , rnasen , tp53 , cox8a , and ccnd1 ) and sox2 in the atc cell line sw1736
. with this purpose in mind , the six interactors together with sox2 were analysed by genemania software   to verify whether their putative network ( figure 1 ) might be enriched in some go annotations . unsurprisingly , the analysis revealed a statistically significant enrichment of mirna - mediated , posttranscriptional gene silencing activities ( table 3 ) .
then we tried to establish in the sw1736 atc cell line whether perturbations in the transcriptional state of sox2 might alter in trans the transcriptional state of the cernas identified .
when we knocked down sox2 transcripts via specific sirna , all the cernas were coherently downregulated in trans in rt - pcr analyses , as expected .
the effect of the downregulation varied from one cerna to another but was always statistically significant   ( figure 2(a ) ) ( table 6 ) . to further investigate whether the effect could be mediated by the impaired transcriptional factor activity of the protein coded by sox2
, we evaluated whether the overexpression of the coding sequence of sox2 could have some in trans effects on cernas .
the coding sequence lacks the 3 untranslated region ( 3utr ) that mainly bears the regulation mediated by mirnas .
our data indicate that no such effect occurs ( figure 2(b ) ) ( table 7 ) , so the trans effect highlighted in ( figure 2(a ) ) is likely to be due to the endogenous mirna competition , as in our hypothesis , rather than a classical interaction mediated by the proteic transcriptional factor sox2 . finally , we investigated whether the overexpression of sox2 3utr might have any effects in trans in the sw1736 atc cell line .
the effects were very modest , if present at all , but in line with the modulation that occurs during the competing events [ 9 , 10 ] .
the most notable effect was the positive correlation , as expected , with the expression of eif2c2 and sox2 itself ( figure 2(c ) ) ( table 8) .
the experiments previously described looked into the effects of perturbation of the expression of sox2 on the expression of cerna genes in an atc cell line .
we then endeavoured to see whether any correlation might exist between the basal expression of sox2 and the cerna genes in different specimens . in detail , we analysed by rt - pcr the relative expression of sox2 and sox2 cernas compared to   -actin expression in sw1736 atc cell line , in 8505c atc cell line , c643 atc cell line , fro atc cell line , bcpap papillary thyroid carcinoma ( ptc ) cell line , tpc-1 ptc cell line , wro follicular thyroid carcinoma , and a pool of normal thyroid tissues present in the laboratory from previous experiments , a pool of limbal stem cells , and isolated lymphocytes from a male donor of 36 years old ( see supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/439370 ) .
interestingly , this analysis suggested a correlation in the basal expression of dicer1 , rnasen , and eif2c2 ( table 9 ) , as can be expected of genes whose functions are strictly coregulated in the biogenesis and function of microrna , but surprisingly their basal expression seemed also to be somehow related to the basal expression of tp53 ( table 9 ) , suggesting interesting scenarios that will be discussed shortly .
the cerna bioinformatics analysis pointed to a list of genes that could be functionally coregulated with the stem transcriptional factor sox2 by a crosstalk mediated by several mirnas . in our analysis , we used interactions reported in the literature instead of bioinformatically predicted ones as done in the past [ 1719 ] .
this approach makes it possible to harvest more solid and reliable data , though it is easier to collect genes that have been previously analysed .
our experiments were pursued in an atc cell line that has previously been demonstrated to constitutively express sox2 that functionally possesses a preeminent hierarchical role on many other stem cell factors , suggesting a leading role in the maintenance of the stemness feature in this cell line .
atc represents a very good candidate for a cancer highly enriched in cscs , which probably are at the core of its unfavourable outcome [ 17 ] . for these reasons
, it is both important to understand the regulatory network that underlies the functions of sox2 in atc , and at the same time an atc cell line is a very good candidate for studying the sox2 network .
looking at the cross - regulation between sox2 and the most probable cernas that we isolated , many if not all the cernas analysed seem to be responsive to alterations in the transcriptional state of sox2 transcripts , independently of the coded protein , suggesting a regulatory network strictly based on noncoding - rnas ( ncrnas ) .
the most striking evidence is the effect of sirna - mediated silencing on sox2 , where all the cernas are accordingly downregulated ( figure 1(a ) ) ( table 6 ) .
by contrast , the overexpression of the sox2 cds alone seems to have almost no effect at all ( figure 1(b ) ) ( table 7 ) .
in contrast , the overexpression of the 3utr of the sox2 transcripts seems to have an upregulation effect in trans , even if not to a great degree ( figure 1(c ) ) ( table 8) .
the 3utr of transcripts is the portion of messengers that is likely to bear the vast majority of regulation mediated by micrornas .
the data reported here are consistent with our hypothesis , so it is reasonable to point to the cernas isolated as potential functional interactors with sox2 , at least in the sw1736 atc cell line .
the interactors isolated pointed to a central role of microrna biogenesis and functions in sox2 activities ( table 3 ) and hence in stemness , as other authors have recently suggested . here
we report that probably the transcription of sox2 stem factors and of dicer ( dicer1 ) , ago2 ( eif2c2 ) , and drosha ( rnasen ) is coregulated by a microrna network . in detail
, drosha is a rna - specific endoribonuclease that is involved in the initial nuclear step of microrna biogenesis .
dicer is a cytoplasmic endoribonuclease that plays a central role in the production of short interfering rnas ( sirna ) and mature micrornas .
sirnas and micrornas serve as a guide to directing the rna - induced silencing complex ( risc ) to complementary rnas to degrade them or prevent their translation .
overall , the mirna pathway is a means to specifically regulate the expression of target genes that seem to directly and indirectly affect tumorigenesis . the sox2 cerna tp53 gene codes for p53 , one of the most studied genes in relation to cancer development .
nevertheless , even a mutated form , if transcribed , can still exert its regulatory functions via its transcript ( e.g. , its 3utr ) . in this perspective , homozygous deletion of the locus or full silencing of the gene perturbs the network differently from a null mutation , which still permits transcription from the locus . in the authors ' opinion
it is interesting to note that some authors have suggested a role of p53 in homoeostasis of the stem niche   and in microrna biogenesis , setting it at a crossroads between cancer , stemness , and microrna biogenesis and functions .
our data are in support of this interpretation , all the more so because the basal transcription of tp53 seems to be correlated with the basal transcription of dicer1 , eif2c2 , and rnasen in the specimens that we analysed , many of them from atc and other thyroid cell lines ( table 9 ) .
the sox2 cerna ccnd1 codes for cyclin d1 , the regulatory subunit that promotes g1/s cell - cycle progression and is involved in oncogenesis .
it has been reported that cyclin d1 induces dicer expression in vitro and in vivo and vice versa and their expression significantly correlates each other ( at least in some subtypes of human breast cancer ) .
it has been suggested that cyclin d1 induction of dicer coordinates microrna biogenesis [ 28 , 35 ] .
our data are in line with the previous results and add a new level of possible crosstalk between dicer1 and ccnd1 , suggesting novel actors in the network previously isolated , such as sox2 or tp53 .
it is likely that cross - regulation between cyclin d1 and dicer might occur in other cancers , especially in atc , which are enriched in sox2 producing cells , which we suggest is part of the network .
the protein encoded by this gene is the terminal enzyme of the respiratory chain that leads to the production of the electrochemical gradient across the inner mitochondrial membrane .
recent discoveries suggest central roles of mitochondria in the maintenance of pluripotency , differentiation , reprogramming , and ageing .
our data suggest possible crosstalk between a crucial nuclear coded mitochondrial factor and cell fate determinants such as sox2 and tp53 .
the sw1736 atc cell line was used to investigate functional sox2 interactors isolated by a novel bioinformatics analysis . because sox2 seems to have a central role in the maintenance of stem features in the sw1736 atc cell line , the interactors are likely to play a role in stemness regulation .
the analysis pointed to dicer1 , eif2c2 , rnasen , tp53 , cox8a , and ccnd1 genes , suggesting that stemness , microrna biogenesis and functions , p53 regulatory network , cyclin d1 , and cell cycle control , together with mitochondrial activity , might be coregulated as a whole in their functions .
our data and previous data from the literature indicate that those functions are strictly interlinked and that deregulation of them might lead to cancer transformation , especially in cancers such as atc that possess an undifferentiated nature suggestive of cancer stem cell enrichment .
 it has been suggested that cancer stem cells ( csc ) may play a central role in oncogenesis , especially in undifferentiated tumours . 
 anaplastic thyroid carcinoma ( atc ) has characteristics suggestive of a tumour enriched in csc . 
 
 previous studies suggested that the stem cell factor sox2 has a preeminent hierarchical role in determining the characteristics of stem cells in sw1736 atc cell line . in detail , silencing sox2 in sw1736 is able to suppress the expression of the stem markers analysed , strongly sensitizing the line to treatment with chemotherapeutic agents . 
 therefore , in order to further investigate the role of sox2 in atc , a competing endogenous rna ( cerna ) analysis was conducted in order to isolate new functional partners of sox2 . among the interactors , of particular interest 
 are genes involved in the biogenesis of mirnas ( dicer1 , rnasen , and eif2c2 ) , in the control cell cycle ( tp53 , ccnd1 ) , and in mitochondrial activity ( cox8a ) . 
 the data suggest that stemness , microrna biogenesis and functions , p53 regulatory network , cyclin d1 , and cell cycle control , together with mitochondrial activity , might be coregulated . 