BACKGROUND
the idea that the greater complexity of higher eukaryotes arises from the portion of the genome called non-coding rnas  is becoming increasingly widespread  <cit> .
indeed, ncrnas are of growing interest, as they have been found to be important regulators of gene expression in development, physiology, and, when dysfunctional, in the presence of disease.
this variegated class of rna species encompasses the well-known mirnas, as well as the most recently acknowledged lncrnas.
discovered first, mirnas have been intensively studied and much is now known about their biological functions, as opposed to lncrnas.
in fact, the latter constitutes a new, potentially fascinating, territory to be explored yet.
mirnas are single-stranded short rnas  that post-transcriptionally regulate gene expression by translation inhibition or degradation of their target mrnas  <cit> .
virtually, all biological processes have been proved to involve mirna regulation, including development, metabolism, cell proliferation, differentiation and apoptosis  <cit> .
accordingly, altered mirna expression characterizes many human diseases and mounting evidence strongly links specific mirnas to tumor initiation, progression and metastasis  <cit> .
several mechanisms, including gene locus amplification  <cit> , chromosomal deletion  <cit> , mutation and epigenetic silencing  <cit> , have been identified as responsible for deregulating mirna expression in cancer.
however, the underlying mechanisms leading to mirna deregulation in cancer are far from being fully understood.
a new mechanism of mirna regulation concerning the ability of rnas to compete for mirna binding has recently been discovered  <cit> .
this intriguing mechanism, also known as ‘target mimicry’ process, was first discovered in plants  <cit> .
ebert et al.
<cit>  later showed that exogenously administered mirna competitors in mammalian cells derepressed mirna targets at least as strongly as chemically modified antisense oligonucleotides.
crucial triggers of this new layer of post-transcriptional regulation are ‘decoys’ - or mirna ‘sponges’ - including both coding and non-coding rnas, such as pseudogenes, large intergenic ncrnas, and circular rnas  <cit> .
sponges exert their decoy activity by recruiting mirna molecules via base-pairing with mirna-recognition elements , which they share with a target, subsequently causing release of the target from mirna control.
poliseno et al.
<cit>  analyzed this mirna removal mechanism by focusing on pseudogenes in samples of both normal and prostate-tumor human tissues.
pseudogenes are defined as copies of real genes that originate from duplications or retro-transpositions.
furthermore, the latter is not translated into functional proteins because their coding potential is corrupted by premature stop codons, deletions/insertions and frameshift mutations.
moving from the evidence that - despite lack of translation - sequence conservation of pseudogenes suggests functionality, the authors proposed them as perfect endogenous competitors of their ancestral genes, because they retain many of the mirna binding sites.
more recently, sumazin et al.
<cit>  investigated the ability of coding and non-coding rnas to act as cernas in human glioblastoma.
they identified a broad network of sponge interactions and suggested them as mediators of crosstalk between different regulatory pathways.
due to the computationally prohibitive burden of testing all possible combination of rna/mirna/rna triplets, the authors only considered those rna/rna pairs sharing a statistically significant number of common mirnas, thus using some a priori information on putative or validated seeds to complementing expression data.
in this paper, we study the role of lncrnas as possible sponge regulators of mirna activity on target mrnas.
we furthermore explored mirna decoy mechanism within gene regulatory circuitry using expression data from tumor and matched normal samples of breast invasive carcinoma , provided by the cancer genome atlas .
our main aim was to probe whether specific lncrnas may function as cernas of protein-coding rnas.
lncrnas are broadly categorized as rnas with more than  <dig> nucleotides lacking an extensive open reading frame  <cit> .
although recent studies have begun to associate subsets of lncrnas to specific regulatory mechanisms  <cit> , the relevance of their role in controlling normal cell physiology and pathogenesis remains unclear.
in our study, we built two networks of lncrna-mrna interactions mediated by mirnas as inferred by multivariate analysis for normal and cancer data, respectively.
the reduced dimensionality of this configuration space, obtained by using a lncrna-centered approach, made the computational burden manageable, with the additional advantage of using a purely data-driven approach.
our study revealed the existence - in normal samples - of a complex regulatory network of mirna-mediated interactions  that appears to be missing in tumor samples.
as a result, an oncosuppressive activity of some specific lncrnas, exploiting a decoy mechanism, is speculated therein.
furthermore, the mmi-network assembled in tumor samples , highlighted some sponge interactions triggered in cancer and shut off in normal tissues, pointing to their potential oncogenic activity.
RESULTS
identification of mirna-mediated mrna/lncrna interactions
we analyzed a large dataset of tumor and matched normal samples of brca profiled for both gene and mirna expression, obtained from tcga.
as discussed in details in the methods section, we restricted our study to a total of  <dig> mrnas,  <dig> mirnas and  <dig> lncrnas.
firstly, we systematically evaluated pearson correlations for all available pairs of  <dig> mrnas and  <dig> mirnas in normal breast and brca samples .
the resulting distribution curves are both unimodal, symmetric and centered at zero, thus not displaying any peculiar underlying correlation pattern.
by contrast, selection for mrnas with at least one co-expressed lncrna  showed the presence, in the normal dataset, of a clear bimodal distribution .
at variance, this effect is not visible using cancer data where the distribution remains unchanged .
these preliminary results suggest a possible involvement of lncrnas in eliciting positive and negative co-expressed mrna/mirna pairs.
repeating the same selection for mrnas with at least one anti-correlated lncrna  yielded the emergence of a similar bimodal behavior .
from a purely statistical framework, positively and negatively correlated mrna/mirna pairs are both interesting.
however, an high positive correlation between rnas competing for mirna binding has been recently experimentally observed and discussed  <cit> .
thus, we focused on mrna/lncrna pairs marked by highly positive correlation - which we called cognate genes in analogy with  <cit>  - to investigate the scenario in which specific mirnas may mediate their interactions .
to pursuit this aim, we applied a well-established tool of multivariate analysis  to each selected mrna/lncrna pair with respect to each mirna in our dataset .
we then computed for each triplet the difference between the pearson and partial correlation coefficients and defined it sensitivitycorrelation :
s=corr-corr.
informally speaking, the partial correlation measures the extent to which an observed correlation between two variables x and y  relies on the presence of a third controlling variable z .
in particular, values of s approaching to zero are indicative of a direct interaction between the two dependent variables , whereas values close to the pearson correlation vaue are indicative of an indirect interaction, suggesting a leading contribution of the explanatory variable .
the sensitivity correlation computed in normal breast samples  unveils an overall trend of mirna-independent interactions between cognate genes  with the notable exception of a limited pool of mirnas .
this marked pattern suggests the existence of specific mirnas, particularly the mir- <dig> family, acting at global level as buffers of mrna/lncrna highly co-expressed pairs.
this finding appeares to be particularly relevant, since it directly points to a limited pool of mirnas capable of establishing a crosstalk throughout the transcriptome as a whole.
computation of the sensitivity correlation repeated in cancer for the same triplets surprisingly resulted in complete disappearance of the observed pattern .
this result pinpoints the presence of a three-way mechanism triggered in normal breast which appears to be shut off in cancer.
interestingly, it may suggest that overriding the interactions of a small group of specific mirnas with their partners - mrnas and lncrnas - could contribute to cancer onset and development.
of note, the high pearson correlation between mrnas and lncrnas that characterizes the top-correlated pairs selected in normal breast  drops when using cancer data.
in fact, the pearson correlation distribution - computed in cancer for the same pairs - becomes nearly symmetric and centered around zero .
the same procedure, i.e.
selection of top-correlated mrna/lncrna pairs followed by computation of sensitivity correlation, was applied to cancer samples .
here, a first difference emerges in the distribution of pearson correlation coefficients, which exhibits a smaller variance and thus a less populated tail of cognate genes .
furthermore, there is lack of evident vertical stripes, despite the presence of sporadic light spots.in the normal dataset, the unimodal and zero-centered distribution of pearson correlation coefficients between all mirnas and all mrnas , when limiting mirnas to that subset which is responsible for the light vertical stripes in the sensitivity correlation heat-map , approaches to a bimodal curve .
this effect seems to be specific for the normal breast since the same mirna selection does not affect the pearson correlation distribution in cancer .to summarize, we observed the emergence of a clear distinction between cancer and normal cells induced by two independent data selection criteria in the mirna/mrna pearson correlation analysis: i. selection based on mrnas having at least one highly correlated lncrna ; ii.
selection based on mirnas mediating mrna/lncrna interactions .
both approaches independently disclosed a tendency towards a bimodal behavior in normal samples, which is unmatched in cancer.
interestingly, when the two selection criteria are adopted together, the bimodal character of the correlation distribution - observed in normal breast - becomes much more evident and witnessed by the sharp breakup of the negative and positive contributions .
again this scenario is unmatched in cancer .
this analysis allowed us to identify two rna-interaction modules that we termed pure sponge module and mixed rna-sponge module.
both of them accommodate mirna-mediated communication between the mrna and the lncrna, but they discriminate the correlation sign linking the mrna and the mirna .
fulfilling cerna features, the pure sponge module may be thought of as a reservoir of putative sponges .
on the contrary, the unexpected positive correlation between the mrna and the mirna in the mixed rna-sponge module may have many different explanations.
among others, we speculate that it may hint to a further layer of regulation ruled by another actor, for instance a transcription factor , which is both a repressor of the mrna and a target of the mirna .
we called this possibility mixed tf-sponge module.
this module may be thought of as a built-in regulatory loop where the transcriptional level is intertwined with the post-transcriptional one.
mirna-mediated interactions networks
in animals, mirnas usually repress the expression of target genes at the post-transcriptional level by binding to partially complementary sites in their 3’ untranslated region .
particularly, watson-crick base-pairing to the seed region, which comprises nucleotides 2- <dig> in the 5’ region of the mature mirna sequence, is important for target recognition  <cit> .
a seed match analysis - ran for each of those mirnas that are responsible of the light vertical stripes in the sensitivity correlation heat-map  - showed that the selected mrna/lncrna pairs are enriched for instances where both rnas harbor one or more binding sites for the related mirna .
integrating the results of multivariate analysis and seed match analysis, we built in normal breast a network of pure and mixed sponge interactions , that we called mirna-mediated interactions network or mmi-network.
nodes in this network represent both mrnas and lnrnas and edges represent mirnas mediating their interactions .
concretely, linked nodes are required to meet two conditions: i. matching high values of the sensitivity correlation ; ii.
harboring one or more mres for the mirnas that they can “sponge”.
we assigned a weight to each mrna/lncrna sponge interaction on the basis of the number of the shared mirnas .
we used the degree of connectivity  of each node in the mmi-network as a suitable parameter of judgement to rank top candidate endogenous decoys for mirnas.
outgoing edges from a lncrna node encompass different types of relationship with the nearest-neighbor mrna nodes.
in particular, a given lncrna can: i. share with the same mrna multiple mirnas for which they compete for binding ; ii.
communicate with several different mrnas through the same mirna ; iii.
communicate with one mrna through a single mirna .
according to this topological measure, we found that the lncrna pvt <dig> with its  <dig> edges represents the first hub in the normal-mmi-network.
it is connected to  <dig> different mrnas  and the mir- <dig> family members are mediating over 80% of these interactions .
notably, the normal mmi-network  is marked by a clear segregation into two internally well connected components: a larger one  mainly dominated by the mir- <dig> family and a smaller one  mainly controlled by mir- <dig>  in particular, we observed an outstanding prevalence of the mir- <dig> family in the whole normal-mmi-network.
in fact, it mediates the most of the communications  between the majority of the lncrnas  and their counterpart mrnas  in the network.
interestingly, the mir- <dig> family members are well-known to be involved in cancer metastasis and are believed to play an essential role in tumor suppression by inhibiting epithelial-to-mesenchymal transition , the initiating step of metastasis  <cit> .
moreover, the mir- <dig> family members have recently been associated to human breast cancer  <cit>  and their overexpression was shown to promote the mesenchymal-to-epithelial transition  <cit> .
here, our analysis suggests that these relevant cancer-associated mirnas hold the reins of communication through the whole mmi-network in normal breast samples.
seeking to functionally explore the two sub-networks evidenced by the above analysis, the lists of protein-coding nodes populating each of them were analyzed for biological functional annotations using the gorilla web tool .
interestingly, the observed topological disjunction seems mirrored by strong enrichment in distinct biological functionalities.
specifically, the larger sub-network is enriched in cell-cell adhesion function , whereas the smaller one is enriched in cellular metabolic processes .
both these biological processes characterize the normal breast epithelium and their proper regulation is essential to maintain tissue integrity.
as for the similarly constructed cancer mmi-network , we observed a clear segregation into two components , yet markedly less populated compared to the normal case.
indeed, the larger sub-network here is composed by  <dig> nodes and  <dig> edges whereas the smaller one is composed by only  <dig> nodes and  <dig> edges .
similarly to the normal case, we assigned a weight to each mrna/lncrna sponge interaction on the basis of the number of the shared mirnas .
in the prevalent component of the cancer mmi-network, mir- <dig> exhibits a leading role by mediating most of the mrna/lncrna connections.
we found that two lncrnas - meg <dig>  and kiaa <dig> - compete for the role of the first hub and regulate the expression of the almost totality of the mrnas in the cancer-mmi-network, by antagonizing mir- <dig> and mir- <dig>  respectively .
of note, meg <dig> was recently suggested to play a significant role as a novel tumor suppressor lncrna in several human cancers and evidence of its association with tumorigenesis is growing every day  <cit> .
functional annotation enrichment analysis of nodes in this larger sub-network clearly points to immune system-related functions .
indeed, inflammation is a hallmark of cancer and different immune cells are known to be involved with either pro- or anti-tumor activity in tumor development  <cit> .
overall, our results indicate that the observed decoy mechanism seems to “switch on” or “off” its agents according to the physiological or pathological condition.
precisely, the framework of mirna-mediated interactions appears completely altered in brca samples compared to normal.
particularly, cerna mechanisms orchestrated by the mir- <dig> family, which is preponderant in the normal breast scenario, disappear in the brca network, where other sponges appear to be activated.
discussion
modes of action in the normal mmi-network
as a case study, we propose two prototypes of pure sponge  and mixed tf-sponge  modules, extracted from the normal breast analysis: the first employs ptenp <dig>  a growth-suppressive lncrna already identified as cerna  <cit> ; the second engages pvt <dig> as a competitor of cdh <dig> for binding to the mir- <dig> family and zeb <dig> as both a transcriptional repressor of cdh <dig> and a target of the mir- <dig> family.
in both cases, the high correlation bridging the mrna/mirna/lncrna actors, which is attained in normal breast , appears completely abolished in breast cancer .
we speculate that the disappearance of the lncrna-mrna crosstalk noticed in cancer may occur because the mirna stops functioning as a mediator of their interaction, with a consequent break-up in the cerna relationship.
in fact, the pearson correlation between cdh <dig> and pvt <dig> is very high in normal breast , but strongly dependent on the mirna, as witnessed by the drastic drop in the correlation after the computational removal of the mirna .
in the example of pure sponge module , ptenp <dig> appears to regulate the expression of hrasls <dig>  a member of the hras-like suppressor family, via antagonizing mir-135b in normal breast tissue.
in humans, hras, together with kras and nras, constitutes the ras protein superfamily that controls proliferation, differentiation and cell cycle via the mitogen-activating kinase  signaling cascade  <cit> .
consistently, we observed this pure sponge module within the smaller sub-network of the normal-mmi-network , which results functionally enriched in the oxidation-reduction process, angiogenesis and regulation of mapk cascade .
these biological processes are closely related.
in fact, the uncontrolled cell growth and division characterizing tumor cells are hampered by a lack of oxygen and other essential nutrients.
to overcome this obstacle, malignant cells acquired the specific ability to induce blood vessel growth , by secreting various growth factors.
recently, reactive oxygen species have also been proposed as essential triggers for angiogenesis  <cit> .
in this light, our observation of a deactivation - in breast cancer - of the sponge mechanism involving ptenp <dig> - together with the previously reported growth-suppressive role of ptenp <dig> <cit>  - corroborates the importance of mirna-mediated ptenp <dig> regulation in cancer  <cit> .
with respect to the mixed tf-sponge module , it is worth noting that the only remaining interaction - found in cancer cells  - is the anti-correlation between mir- <dig> and its validated target zeb <dig> <cit> .
moreover, the relationship between zeb <dig> and cdh <dig>  hypothesized by our analysis, has been experimentally validated  <cit> , corroborating the relevance of our findings.
specifically, zeb <dig> results downregulated in our breast cancer dataset compared to normal samples, while both the mir- <dig> family members and cdh <dig> are overexpressed.
this is suggestive of an epithelial-like phenotype maintained by high levels of the mir- <dig> family members, which inhibit zeb <dig> and, hence, increases the expression of zeb-repressed epithelial genes, such as cdh <dig>   <cit> .
however, it has been shown that zeb <dig> triggers a double negative feedforward loop, by downregulating its own inhibitors   <cit> .
thus, depending on the zeb <dig> levels in cancer cells, this loop could stabilize either mesenchymal or epithelial differentiation, accounting for the phenotypic heterogeneity viewed in tumors and metastases.
in particular, the switch to a mesenchymal state can be induced by the transforming growth factor tgf β, which increases zeb <dig>  while ectopic expression of the mir- <dig> family members, which reduces zeb <dig>  seems either to prevent tgf β-induced emt or to initiate epithelial-like reversion in mesenchymal cells  <cit> .
pvt <dig> is the main cerna regulator in normal breast
pvt <dig> is a lncrna that appears to be strongly conserved between mouse and human  <cit>  and amplification of its locus is one of the most frequent events in breast cancer  <cit> .
moreover, its overexpression has been recently suggested to contribute to breast pathogenesis by inhibiting apoptosis  <cit> .
we found that pvt <dig> acts as cerna in the normal-mmi-network, but not in cancer.
moreover, it reveals a net binding preference towards the mir- <dig> family , which it antagonizes to regulate the expression of hundreds of mrnas in the normal case.
in terms of topological properties, pvt <dig> switches from being the first of the hubs in the normal-mmi-network to fall outside the list of nodes of the cancer network.
interestingly, recent studies suggested a role for pvt <dig> in the pathophysiology of breast cancer by virtue of pvt1-mediated inhibition of apoptosis, when overexpressed  <cit> .
among pvt <dig> nearest-neighbors in the normal-mmi-network, we emphasize some important regulators of breast tissue morphogenesis and development, such as: the aforementioned cdh1; all three members of the extended p <dig> family ; two members of the mammalian runx family  and gata <dig> .
tp <dig> is the most extensively studied tumor suppressor and acts in response to diverse forms of cellular stresses to induce cell cycle arrest, apoptosis and senescence  <cit> .
the two identified homologues, tp <dig> and tp <dig>  have also been related to apoptosis, and a possible role as tumor suppressors has been suggested  <cit> .
runx <dig> is the predominant runx family member expressed in human breast epithelial cells and there is a growing body of evidence suggesting its possible role as a breast cancer suppressor  <cit> .
runx <dig> has been recently reviewed as a tumor suppressor, specifically in human brca, with decreasing expression associated to disease progression  <cit> .
finally, gata <dig> has been linked to mammary gland morphogenesis, mammary tumor differentiation and metastasis  <cit> .
in sum: i. the pvt <dig> neighborhood in the normal-mmi-network encompasses cancer related genes as well as genes involved in mammary gland development and cell morphogenesis; and ii.
the sponge program orchestrated by pvt <dig> results completely abolished in cancer.
taken together these findings may be indicative of a possible pvt <dig> surveillance role aimed to preserve cell-cell adhesion.
indeed, mammary gland morphogenesis results from the coordination of diverse cellular processes involving cell-cell adhesion, migration, proliferation and apoptosis.
thus, pvt <dig> controlling circuit may provide further insight in solving this complicated puzzle.
the pvt <dig> sponge program is turned off in breast cancer
the specific conditions required for a cerna network to occur are still far from being determined.
the importance of the relative concentration of the cernas, and their related mirnas, has been recently emphasized  <cit> .
in fact, in their study, salmena et al.
<cit> , suggest that large changes in the cerna expression levels either overcome, or relieve, the mirna repression on competing cernas; similarly, a very large mirna overexpression may abolish competition.
along this line, the mir- <dig> family members appear highly upregulated in the cancer dataset that we analyzed .
this may explain the observed annihilation of the sponge interactions that they mediate.
however, this model may be undermined by the evidence that pvt <dig> - the main sponge regulator of the mir- <dig> family in the normal network - also results upregulated  in cancer.
examination of the pvt <dig> genomic locus showed the existence of multiple isoforms, whose sequence analysis prompted us to formulate an alternative hypothesis.
in particular, members of the mir- <dig> family can be grouped in two clusters based on the seed sequence , differing by one nucleotide.
despite most of the pvt <dig> alternative isoforms harboring seed matches for both clusters , two isoforms lack the putative mres for the mir- <dig> family.
this is the result of alternative splicing events as well as the presence of alternative transcription start sites that cause skipping of the exons where the mres reside.
hence, the observed withdrawal in cancer of the pvt <dig> sponge activity may be due to preferential expression of these two isoforms, independently from the abundance of pvt <dig>
comparison between mmi-network and correlation network
we compared the mmi-networks with their cognate correlation networks , composed by highly correlated mrna/lncrna pairs .
interestingly, we noticed that the shape of both normal and cancer-mmi-networks is reminiscent of their ancestor correlation networks.
in particular, the two components, which characterize the normal-mmi-network , emerge as already separated, when mapped onto the corresponding correlation network .
in fact, the correlation network itself appears to be weakly connected and a tendency to split into two sub-regions is already conceivable .
on the other hand, the cancer correlation network is clearly composed by a giant highly tangled component  that mirrors the single prevalent sub-network dominating the cancer-mmi-network, disregarding the limited residual component .
taken together, the analyses of the correlation and mmi-networks strengthen the hypothesis that the normal breast is marked by a more structured organization as opposed to the disordered malignant picture.
CONCLUSIONS
we propose a novel computational approach suitable to exploring the potential role of lncrnas as cernas.
we applied our method to a large dataset of brca obtained from tcga and built two networks of cerna interactions in normal and cancerous state.
overall, we noticed a dramatic difference between the physiological and pathological condition concerning the identification and the amount of activated sponges.
the drastic change observed in the sponge program is suggestive of a marked cerna rewiring that characterizes the cancer state.
the main actor of this “system-reset” is pvt <dig>  despite its upregulation, it stops working as cerna in the cancerous state.
we speculate that the withdrawal in cancer of the pvt <dig> cerna activity can be due to the preferential expression of the two isoforms missing the binding sites for the mir- <dig> family.

 the idea that the greater complexity of higher eukaryotes arises from the portion of the genome called non-coding rnas is becoming increasingly widespread. indeed, ncrnas are of growing interest, as they have been found to be important regulators of gene expression in development, physiology, and, when dysfunctional, in the presence of disease. this variegated class of rna species encompasses the well-known mirnas, as well as the most recently acknowledged lncrnas. discovered first, mirnas have been intensively studied and much is now known about their biological functions, as opposed to lncrnas. in fact, the latter constitutes a new, potentially fascinating, territory to be explored yet. mirnas are single-stranded short rnas that post-transcriptionally regulate gene expression by translation inhibition or degradation of their target mrnas. virtually, all biological processes have been proved to involve mirna regulation, including development, metabolism, cell proliferation, differentiation and apoptosis. accordingly, altered mirna expression characterizes many human diseases and mounting evidence strongly links specific mirnas to tumor initiation, progression and metastasis. several mechanisms, including gene locus amplification, chromosomal deletion, mutation and epigenetic silencing, have been identified as responsible for deregulating mirna expression in cancer. however, the underlying mechanisms leading to mirna deregulation in cancer are far from being fully understood. a new mechanism of mirna regulation concerning the ability of rnas to compete for mirna binding has recently been discovered. this intriguing mechanism, also known as ‘target mimicry’ process, was first discovered in plants. ebert et al. later showed that exogenously administered mirna competitors in mammalian cells derepressed mirna targets at least as strongly as chemically modified antisense oligonucleotides. crucial triggers of this new layer of post-transcriptional regulation are ‘decoys’ - or mirna ‘sponges’ - including both coding and non-coding rnas, such as pseudogenes, large intergenic ncrnas, and circular rnas. sponges exert their decoy activity by recruiting mirna molecules via base-pairing with mirna-recognition elements, which they share with a target, subsequently causing release of the target from mirna control. poliseno et al. analyzed this mirna removal mechanism by focusing on pseudogenes in samples of both normal and prostate-tumor human tissues. pseudogenes are defined as copies of real genes that originate from duplications or retro-transpositions. furthermore, the latter is not translated into functional proteins because their coding potential is corrupted by premature stop codons, deletions/insertions and frameshift mutations. moving from the evidence that - despite lack of translation - sequence conservation of pseudogenes suggests functionality, the authors proposed them as perfect endogenous competitors of their ancestral genes, because they retain many of the mirna binding sites. more recently, sumazin et al. investigated the ability of coding and non-coding rnas to act as cernas in human glioblastoma. they identified a broad network of sponge interactions and suggested them as mediators of crosstalk between different regulatory pathways. due to the computationally prohibitive burden of testing all possible combination of rna/mirna/rna triplets, the authors only considered those rna/rna pairs sharing a statistically significant number of common mirnas, thus using some a priori information on putative or validated seeds to complementing expression data. in this paper, we study the role of lncrnas as possible sponge regulators of mirna activity on target mrnas. we furthermore explored mirna decoy mechanism within gene regulatory circuitry using expression data from tumor and matched normal samples of breast invasive carcinoma, provided by the cancer genome atlas. our main aim was to probe whether specific lncrnas may function as cernas of protein-coding rnas. lncrnas are broadly categorized as rnas with more than nucleotides lacking an extensive open reading frame. although recent studies have begun to associate subsets of lncrnas to specific regulatory mechanisms, the relevance of their role in controlling normal cell physiology and pathogenesis remains unclear. in our study, we built two networks of lncrna-mrna interactions mediated by mirnas as inferred by multivariate analysis for normal and cancer data, respectively. the reduced dimensionality of this configuration space, obtained by using a lncrna-centered approach, made the computational burden manageable, with the additional advantage of using a purely data-driven approach. our study revealed the existence - in normal samples - of a complex regulatory network of mirna-mediated interactions that appears to be missing in tumor samples. as a result, an oncosuppressive activity of some specific lncrnas, exploiting a decoy mechanism, is speculated therein. furthermore, the mmi-network assembled in tumor samples, highlighted some sponge interactions triggered in cancer and shut off in normal tissues, pointing to their potential oncogenic activity.  identification of mirna-mediated mrna/lncrna interactions we analyzed a large dataset of tumor and matched normal samples of brca profiled for both gene and mirna expression, obtained from tcga. as discussed in details in the methods section, we restricted our study to a total of mrnas, mirnas and lncrnas. firstly, we systematically evaluated pearson correlations for all available pairs of mrnas and mirnas in normal breast and brca samples. the resulting distribution curves are both unimodal, symmetric and centered at zero, thus not displaying any peculiar underlying correlation pattern. by contrast, selection for mrnas with at least one co-expressed lncrna showed the presence, in the normal dataset, of a clear bimodal distribution. at variance, this effect is not visible using cancer data where the distribution remains unchanged. these preliminary results suggest a possible involvement of lncrnas in eliciting positive and negative co-expressed mrna/mirna pairs. repeating the same selection for mrnas with at least one anti-correlated lncrna yielded the emergence of a similar bimodal behavior. from a purely statistical framework, positively and negatively correlated mrna/mirna pairs are both interesting. however, an high positive correlation between rnas competing for mirna binding has been recently experimentally observed and discussed. thus, we focused on mrna/lncrna pairs marked by highly positive correlation - which we called cognate genes in analogy with - to investigate the scenario in which specific mirnas may mediate their interactions. to pursuit this aim, we applied a well-established tool of multivariate analysis to each selected mrna/lncrna pair with respect to each mirna in our dataset. we then computed for each triplet the difference between the pearson and partial correlation coefficients and defined it sensitivitycorrelation: s=corr-corr. informally speaking, the partial correlation measures the extent to which an observed correlation between two variables x and y relies on the presence of a third controlling variable z. in particular, values of s approaching to zero are indicative of a direct interaction between the two dependent variables, whereas values close to the pearson correlation vaue are indicative of an indirect interaction, suggesting a leading contribution of the explanatory variable. the sensitivity correlation computed in normal breast samples unveils an overall trend of mirna-independent interactions between cognate genes with the notable exception of a limited pool of mirnas. this marked pattern suggests the existence of specific mirnas, particularly the mir- family, acting at global level as buffers of mrna/lncrna highly co-expressed pairs. this finding appeares to be particularly relevant, since it directly points to a limited pool of mirnas capable of establishing a crosstalk throughout the transcriptome as a whole. computation of the sensitivity correlation repeated in cancer for the same triplets surprisingly resulted in complete disappearance of the observed pattern. this result pinpoints the presence of a three-way mechanism triggered in normal breast which appears to be shut off in cancer. interestingly, it may suggest that overriding the interactions of a small group of specific mirnas with their partners - mrnas and lncrnas - could contribute to cancer onset and development. of note, the high pearson correlation between mrnas and lncrnas that characterizes the top-correlated pairs selected in normal breast drops when using cancer data. in fact, the pearson correlation distribution - computed in cancer for the same pairs - becomes nearly symmetric and centered around zero. the same procedure, i.e. selection of top-correlated mrna/lncrna pairs followed by computation of sensitivity correlation, was applied to cancer samples. here, a first difference emerges in the distribution of pearson correlation coefficients, which exhibits a smaller variance and thus a less populated tail of cognate genes. furthermore, there is lack of evident vertical stripes, despite the presence of sporadic light spots.in the normal dataset, the unimodal and zero-centered distribution of pearson correlation coefficients between all mirnas and all mrnas, when limiting mirnas to that subset which is responsible for the light vertical stripes in the sensitivity correlation heat-map, approaches to a bimodal curve. this effect seems to be specific for the normal breast since the same mirna selection does not affect the pearson correlation distribution in cancer.to summarize, we observed the emergence of a clear distinction between cancer and normal cells induced by two independent data selection criteria in the mirna/mrna pearson correlation analysis: i. selection based on mrnas having at least one highly correlated lncrna; ii. selection based on mirnas mediating mrna/lncrna interactions. both approaches independently disclosed a tendency towards a bimodal behavior in normal samples, which is unmatched in cancer. interestingly, when the two selection criteria are adopted together, the bimodal character of the correlation distribution - observed in normal breast - becomes much more evident and witnessed by the sharp breakup of the negative and positive contributions. again this scenario is unmatched in cancer. this analysis allowed us to identify two rna-interaction modules that we termed pure sponge module and mixed rna-sponge module. both of them accommodate mirna-mediated communication between the mrna and the lncrna, but they discriminate the correlation sign linking the mrna and the mirna. fulfilling cerna features, the pure sponge module may be thought of as a reservoir of putative sponges. on the contrary, the unexpected positive correlation between the mrna and the mirna in the mixed rna-sponge module may have many different explanations. among others, we speculate that it may hint to a further layer of regulation ruled by another actor, for instance a transcription factor, which is both a repressor of the mrna and a target of the mirna. we called this possibility mixed tf-sponge module. this module may be thought of as a built-in regulatory loop where the transcriptional level is intertwined with the post-transcriptional one. mirna-mediated interactions networks in animals, mirnas usually repress the expression of target genes at the post-transcriptional level by binding to partially complementary sites in their 3’ untranslated region. particularly, watson-crick base-pairing to the seed region, which comprises nucleotides 2- in the 5’ region of the mature mirna sequence, is important for target recognition. a seed match analysis - ran for each of those mirnas that are responsible of the light vertical stripes in the sensitivity correlation heat-map - showed that the selected mrna/lncrna pairs are enriched for instances where both rnas harbor one or more binding sites for the related mirna. integrating the results of multivariate analysis and seed match analysis, we built in normal breast a network of pure and mixed sponge interactions, that we called mirna-mediated interactions network or mmi-network. nodes in this network represent both mrnas and lnrnas and edges represent mirnas mediating their interactions. concretely, linked nodes are required to meet two conditions: i. matching high values of the sensitivity correlation; ii. harboring one or more mres for the mirnas that they can “sponge”. we assigned a weight to each mrna/lncrna sponge interaction on the basis of the number of the shared mirnas. we used the degree of connectivity of each node in the mmi-network as a suitable parameter of judgement to rank top candidate endogenous decoys for mirnas. outgoing edges from a lncrna node encompass different types of relationship with the nearest-neighbor mrna nodes. in particular, a given lncrna can: i. share with the same mrna multiple mirnas for which they compete for binding; ii. communicate with several different mrnas through the same mirna; iii. communicate with one mrna through a single mirna. according to this topological measure, we found that the lncrna pvt with its edges represents the first hub in the normal-mmi-network. it is connected to different mrnas and the mir- family members are mediating over 80% of these interactions. notably, the normal mmi-network is marked by a clear segregation into two internally well connected components: a larger one mainly dominated by the mir- family and a smaller one mainly controlled by mir- in particular, we observed an outstanding prevalence of the mir- family in the whole normal-mmi-network. in fact, it mediates the most of the communications between the majority of the lncrnas and their counterpart mrnas in the network. interestingly, the mir- family members are well-known to be involved in cancer metastasis and are believed to play an essential role in tumor suppression by inhibiting epithelial-to-mesenchymal transition, the initiating step of metastasis. moreover, the mir- family members have recently been associated to human breast cancer and their overexpression was shown to promote the mesenchymal-to-epithelial transition. here, our analysis suggests that these relevant cancer-associated mirnas hold the reins of communication through the whole mmi-network in normal breast samples. seeking to functionally explore the two sub-networks evidenced by the above analysis, the lists of protein-coding nodes populating each of them were analyzed for biological functional annotations using the gorilla web tool. interestingly, the observed topological disjunction seems mirrored by strong enrichment in distinct biological functionalities. specifically, the larger sub-network is enriched in cell-cell adhesion function, whereas the smaller one is enriched in cellular metabolic processes. both these biological processes characterize the normal breast epithelium and their proper regulation is essential to maintain tissue integrity. as for the similarly constructed cancer mmi-network, we observed a clear segregation into two components, yet markedly less populated compared to the normal case. indeed, the larger sub-network here is composed by nodes and edges whereas the smaller one is composed by only nodes and edges. similarly to the normal case, we assigned a weight to each mrna/lncrna sponge interaction on the basis of the number of the shared mirnas. in the prevalent component of the cancer mmi-network, mir- exhibits a leading role by mediating most of the mrna/lncrna connections. we found that two lncrnas - meg and kiaa - compete for the role of the first hub and regulate the expression of the almost totality of the mrnas in the cancer-mmi-network, by antagonizing mir- and mir- respectively. of note, meg was recently suggested to play a significant role as a novel tumor suppressor lncrna in several human cancers and evidence of its association with tumorigenesis is growing every day. functional annotation enrichment analysis of nodes in this larger sub-network clearly points to immune system-related functions. indeed, inflammation is a hallmark of cancer and different immune cells are known to be involved with either pro- or anti-tumor activity in tumor development. overall, our results indicate that the observed decoy mechanism seems to “switch on” or “off” its agents according to the physiological or pathological condition. precisely, the framework of mirna-mediated interactions appears completely altered in brca samples compared to normal. particularly, cerna mechanisms orchestrated by the mir- family, which is preponderant in the normal breast scenario, disappear in the brca network, where other sponges appear to be activated. discussion modes of action in the normal mmi-network as a case study, we propose two prototypes of pure sponge and mixed tf-sponge modules, extracted from the normal breast analysis: the first employs ptenp a growth-suppressive lncrna already identified as cerna; the second engages pvt as a competitor of cdh for binding to the mir- family and zeb as both a transcriptional repressor of cdh and a target of the mir- family. in both cases, the high correlation bridging the mrna/mirna/lncrna actors, which is attained in normal breast, appears completely abolished in breast cancer. we speculate that the disappearance of the lncrna-mrna crosstalk noticed in cancer may occur because the mirna stops functioning as a mediator of their interaction, with a consequent break-up in the cerna relationship. in fact, the pearson correlation between cdh and pvt is very high in normal breast, but strongly dependent on the mirna, as witnessed by the drastic drop in the correlation after the computational removal of the mirna. in the example of pure sponge module, ptenp appears to regulate the expression of hrasls a member of the hras-like suppressor family, via antagonizing mir-135b in normal breast tissue. in humans, hras, together with kras and nras, constitutes the ras protein superfamily that controls proliferation, differentiation and cell cycle via the mitogen-activating kinase signaling cascade. consistently, we observed this pure sponge module within the smaller sub-network of the normal-mmi-network, which results functionally enriched in the oxidation-reduction process, angiogenesis and regulation of mapk cascade. these biological processes are closely related. in fact, the uncontrolled cell growth and division characterizing tumor cells are hampered by a lack of oxygen and other essential nutrients. to overcome this obstacle, malignant cells acquired the specific ability to induce blood vessel growth, by secreting various growth factors. recently, reactive oxygen species have also been proposed as essential triggers for angiogenesis. in this light, our observation of a deactivation - in breast cancer - of the sponge mechanism involving ptenp - together with the previously reported growth-suppressive role of ptenp - corroborates the importance of mirna-mediated ptenp regulation in cancer. with respect to the mixed tf-sponge module, it is worth noting that the only remaining interaction - found in cancer cells - is the anti-correlation between mir- and its validated target zeb. moreover, the relationship between zeb and cdh hypothesized by our analysis, has been experimentally validated, corroborating the relevance of our findings. specifically, zeb results downregulated in our breast cancer dataset compared to normal samples, while both the mir- family members and cdh are overexpressed. this is suggestive of an epithelial-like phenotype maintained by high levels of the mir- family members, which inhibit zeb and, hence, increases the expression of zeb-repressed epithelial genes, such as cdh. however, it has been shown that zeb triggers a double negative feedforward loop, by downregulating its own inhibitors. thus, depending on the zeb levels in cancer cells, this loop could stabilize either mesenchymal or epithelial differentiation, accounting for the phenotypic heterogeneity viewed in tumors and metastases. in particular, the switch to a mesenchymal state can be induced by the transforming growth factor tgf β, which increases zeb while ectopic expression of the mir- family members, which reduces zeb seems either to prevent tgf β-induced emt or to initiate epithelial-like reversion in mesenchymal cells. pvt is the main cerna regulator in normal breast pvt is a lncrna that appears to be strongly conserved between mouse and human and amplification of its locus is one of the most frequent events in breast cancer. moreover, its overexpression has been recently suggested to contribute to breast pathogenesis by inhibiting apoptosis. we found that pvt acts as cerna in the normal-mmi-network, but not in cancer. moreover, it reveals a net binding preference towards the mir- family, which it antagonizes to regulate the expression of hundreds of mrnas in the normal case. in terms of topological properties, pvt switches from being the first of the hubs in the normal-mmi-network to fall outside the list of nodes of the cancer network. interestingly, recent studies suggested a role for pvt in the pathophysiology of breast cancer by virtue of pvt1-mediated inhibition of apoptosis, when overexpressed. among pvt nearest-neighbors in the normal-mmi-network, we emphasize some important regulators of breast tissue morphogenesis and development, such as: the aforementioned cdh1; all three members of the extended p family; two members of the mammalian runx family and gata. tp is the most extensively studied tumor suppressor and acts in response to diverse forms of cellular stresses to induce cell cycle arrest, apoptosis and senescence. the two identified homologues, tp and tp have also been related to apoptosis, and a possible role as tumor suppressors has been suggested. runx is the predominant runx family member expressed in human breast epithelial cells and there is a growing body of evidence suggesting its possible role as a breast cancer suppressor. runx has been recently reviewed as a tumor suppressor, specifically in human brca, with decreasing expression associated to disease progression. finally, gata has been linked to mammary gland morphogenesis, mammary tumor differentiation and metastasis. in sum: i. the pvt neighborhood in the normal-mmi-network encompasses cancer related genes as well as genes involved in mammary gland development and cell morphogenesis; and ii. the sponge program orchestrated by pvt results completely abolished in cancer. taken together these findings may be indicative of a possible pvt surveillance role aimed to preserve cell-cell adhesion. indeed, mammary gland morphogenesis results from the coordination of diverse cellular processes involving cell-cell adhesion, migration, proliferation and apoptosis. thus, pvt controlling circuit may provide further insight in solving this complicated puzzle. the pvt sponge program is turned off in breast cancer the specific conditions required for a cerna network to occur are still far from being determined. the importance of the relative concentration of the cernas, and their related mirnas, has been recently emphasized. in fact, in their study, salmena et al., suggest that large changes in the cerna expression levels either overcome, or relieve, the mirna repression on competing cernas; similarly, a very large mirna overexpression may abolish competition. along this line, the mir- family members appear highly upregulated in the cancer dataset that we analyzed. this may explain the observed annihilation of the sponge interactions that they mediate. however, this model may be undermined by the evidence that pvt - the main sponge regulator of the mir- family in the normal network - also results upregulated in cancer. examination of the pvt genomic locus showed the existence of multiple isoforms, whose sequence analysis prompted us to formulate an alternative hypothesis. in particular, members of the mir- family can be grouped in two clusters based on the seed sequence, differing by one nucleotide. despite most of the pvt alternative isoforms harboring seed matches for both clusters, two isoforms lack the putative mres for the mir- family. this is the result of alternative splicing events as well as the presence of alternative transcription start sites that cause skipping of the exons where the mres reside. hence, the observed withdrawal in cancer of the pvt sponge activity may be due to preferential expression of these two isoforms, independently from the abundance of pvt comparison between mmi-network and correlation network we compared the mmi-networks with their cognate correlation networks, composed by highly correlated mrna/lncrna pairs. interestingly, we noticed that the shape of both normal and cancer-mmi-networks is reminiscent of their ancestor correlation networks. in particular, the two components, which characterize the normal-mmi-network, emerge as already separated, when mapped onto the corresponding correlation network. in fact, the correlation network itself appears to be weakly connected and a tendency to split into two sub-regions is already conceivable. on the other hand, the cancer correlation network is clearly composed by a giant highly tangled component that mirrors the single prevalent sub-network dominating the cancer-mmi-network, disregarding the limited residual component. taken together, the analyses of the correlation and mmi-networks strengthen the hypothesis that the normal breast is marked by a more structured organization as opposed to the disordered malignant picture.  we propose a novel computational approach suitable to exploring the potential role of lncrnas as cernas. we applied our method to a large dataset of brca obtained from tcga and built two networks of cerna interactions in normal and cancerous state. overall, we noticed a dramatic difference between the physiological and pathological condition concerning the identification and the amount of activated sponges. the drastic change observed in the sponge program is suggestive of a marked cerna rewiring that characterizes the cancer state. the main actor of this “system-reset” is pvt despite its upregulation, it stops working as cerna in the cancerous state. we speculate that the withdrawal in cancer of the pvt cerna activity can be due to the preferential expression of the two isoforms missing the binding sites for the mir- family. 
this variegated class of rna species encompasses the well-known micrornas and the most recently acknowledged long non-coding rnas. we built normal and cancer networks of mirna-mediated sponge interactions using breast cancer expression data provided by the cancer genome atlas. at the heart of this phenomenon is the lncrna pvt as illustrated by both the width of its antagonist mrnas in normal-mmi-network, and the relevance of the latter in breast cancer. we propose a computational approach to explore the ability of lncrnas to act as cernas by protecting mrnas from mirna repression.
 non-coding rnas are emerging as key regulators of many cellular processes in both physiological and pathological states. moreover, the constant discovery of new non-coding rna species suggests that the study of their complex functions is still in its very early stages. this variegated class of rna species encompasses the well-known micrornas and the most recently acknowledged long non-coding rnas. interestingly, in the last couple of years, a few studies have shown that some lncrnas can act as mirna sponges, i.e. as competing endogenous rnas, able to reduce the amount of mirnas available to target messenger rnas.  we propose a computational approach to explore the ability of lncrnas to act as cernas by protecting mrnas from mirna repression. a seed match analysis was performed to validate the underlying regression model. we built normal and cancer networks of mirna-mediated sponge interactions using breast cancer expression data provided by the cancer genome atlas.  our study highlights a marked rewiring in the cerna program between normal and pathological breast tissue, documented by its “on/off” switch from normal to cancer, and vice-versa. this mutually exclusive activation confers an interesting character to cernas as potential oncosuppressive, or oncogenic, protagonists in cancer. at the heart of this phenomenon is the lncrna pvt as illustrated by both the width of its antagonist mrnas in normal-mmi-network, and the relevance of the latter in breast cancer. interestingly, pvt revealed a net binding preference towards the mir- family as the bone of contention with its rival mrnas. systems biologynetworks analysisepigenetics 