annually more than a million new diagnoses worldwide for breast cancer ( bc ) emerge , becoming the most common female malignancy .
the term  breast cancer  refers to a complex disease , displaying distinctive histologic and genetic features .
dramatic changes occur in gene expression during the transformation of normal breast tissues into ductal in situ carcinoma ( dcis ) , much less important , and somewhat surprisingly , in the transition from dcis to invasive ductal carcinoma ( idc ) .
in fact , those from the same histological subtype share very similar genetic , epigenetic alterations and gene expression patterns .
dcis and matched adjacent idc ( synchronous dcis and idc ) have remarkably similar genetic and copy number profile [ 2 , 3 ] .
therefore , none of the current biomarkers , or genomic classifications , can predict if , and when , dcis will progress to idc . in principle , a better understanding of dcis progression could highlight ways to arrest invasion at the preinvasive stage . from coding to noncoding , high - throughput next generation sequencing ( ngs ) has been analyzing in depth the entire genome at base - pair resolution .
the noncoding panorama is a real opportunity to reveal new biomarkers or potential targets : in this landscape long noncoding rnas ( lncrnas ) are an emerging class of key regulatory rnas that do not code for protein and are not translated .
lncrnas are crucial players in various key biological processes that include dosage compensation , genomic imprinting , chromatin organization , gene regulation , and alternative splicing .
more recently , the research interest is focused on circular rnas ( circrnas ) , a large class of long noncoding rnas circularized and discovered from a handful of transcribed genes more than twenty years ago .
nowadays , there are different types of circrnas   and are known to be abundant , conserved , and stable in cytoplasm and even in blood . at the time of the discovery these rna molecules had generally been considered to be of low abundance and likely representing errors in splicing .
circrnas are nontranslated rnas , typically nonpolyadenylated , with a resistance to exonucleases that gives them the ability to be more stable than the common linear rna isoforms , also increasing proper folding by imposing structural constraints on the rna .
their formation is due to a non - random back - splice event that involves in a covalent junction between the 3 and 5 ends , providing covalently closed continuous loops configuration , in opposition to the theory that for decades considering the circrnas as the result of gene rearrangements or splicing artifacts producing rare isoforms [ 10 , 11 ] .
several studies revealed that circrnas are usually composed of 1 to 5 exons , where each exon can be up to three times longer than the average expressed exon .
this perhaps suggests that the probability to circularizing for an exon is directly proportional to the length of that exon .
although the formation mechanism and the cellular function have been completely understood , the function and the expressions are not clear .
the majority of circrnas often exhibit tissue / developmental - stage - specific expression , described recently for the brain .
circrnas are also deregulated in cancer cell lines , but not many studies have been performed yet on cancer patients . recently , some studies have been providing evidences about the interaction between circrna and mirna , supporting a  sponge effect  of the circrnas as posttranscriptional regulators of other ncrnas , such as mirnas [ 10 , 14 ] . based on these findings , we will define the panel of expressed circrnas , in dcis and idc samples .
we predicted strong circrnas candidates , enforcing their existence with an independent false discovery polyadenylated subtraction , and then we studied the possible roles of selected circrnas .
publicly available rna - seq data of total rna from ten tandem dcis / idc samples from five patients afflicted with dcis that are synchronous with idc within the same breast ( gse66301 , downloaded from the sequence read archive ( sra ) ) ( illumina gaii , paired - end sequencing ( 2  75  nt length ) ) .
gse66301 series reports data from 6 pairs of dcis - idc samples , we excluded one pair that has one sample that not respected the high media level ( > 88% ) of the mapping quality reads percentage , showing the 49% .
reference genome human hg19 ( february 2009 , grch37 ) , was downloaded from the ucsc genome browser ( http://genome.ucsc.edu/ ) . for circexplorer we used star as aligner software .
we also used the fastq.gz samples of the mcf7 cell line . these are public and available at the encode repository site ( http://hgdownload.cse.ucsc.edu/goldenpath/hg19/encodedcc/ ) : poly(a ) and nonpoly(a ) selected rna - seq data , generated with paired - end sequencing ( 2  75  nt length ) , after the trimming quality control steps . for circrnas ,
the raw read counts were normalized to sequencing depth by dividing the raw number with the number of total reads mapped to and multiply for 10 ( srpbm : reads per billion mapped reads ) .
rpm ( reads per million ) was used for the mrnas . the circbase track on genome browser
was consulted to verify the annotation and if the circrnas was showed by other previous experiments . for the enrichment analysis of the potential disease involved with expression of circrnas we used the circ2trait tool .
mirtarbase ( http://mirtarbase.mbc.nctu.edu.tw/ ) was used to find the genes involved in emt and targeted by the human mir-200b , mir-200c , and mir.429 .
custom pipelines to identify : different and common circrnas , and to create graphics , were performed using r software ( http://www.r-project.org/ ) .
the raw total rna sequencing data , after quality control procedures ( trimming ) , was used for running the bioinformatic detection pipeline : circexplorer .
we used this algorithm because it was one of the mostly used and cited in many sounded scientific research ; and in comparison to other four pipelines , it has shown high accuracy and a good sensitivity . briefly
, circexplorer was focused on the identification of junction reads from back - spliced exons and intron lariats ( the terminal parts of all unmapped reads that are aligned independently to the genome ) . in parallel , we analyzed circular rnas expression of the total rna - seq of mcf7 , an invasive breast ductal carcinoma cell line .
this analysis revealed us the candidates circular rnas expressed in polyadenylated ( polya+ ) and not polya+ selected samples .
previous studies have reported that circrnas were nonpolyadenylated   or rnase r - resistant , then we expected that the circrnas discovered using poly(a)+ selection could be the results of miscellaneous amplification products , misalignment on references genomes or false positives , probably not well filtered by the quality control software .
all the circrnas , predicted in the polya+ samples , were considered false positive and then used as criteria of exclusion .
basically , we applied a false discovery polyadenylated subtraction from the output lists of all the circrnas candidates of each samples .
following these selection criteria this procedure would gave us a list of high - confidence circrnas .
here we listed an example of the command parameters used for  circexplorer pipeline  : //alignment star -  -genomedir 
  fastq2  -  -readfilescommand zcat -  -runthreadn 8 -  -chimsegmentmin 20 -  -chimjunctionoverhangmin 20 -  -outfilenameprefix $ starout_dir 1log_file  2err_file  //convert chimeric to file.txt./star_parse.py chimeric.out.junction fusion_junction.txt //search circrnas./circexplorer.py -j fusion_junction.txt -g  genome.fa 
dcis invasive progression has been characterized by investigating the circrnas expression in tandem dcis / idc model using five samples patients afflicted with dcis that were synchronous with idc within the same breast ( gse66301 ) .
we obtained for each sample the list of circrnas candidates from algorithms ' output and refined by the false positive subtraction ( see section 2 for details ) . as expected , there were differences in terms of the total amount of circrnas identified in each sample : the circexplorer found 1938 raw circrnas with one read .
the total candidate distinct circular rnas that passed our criteria were 756 in the total of the breast cancer : 552 expressed in dcis and 208 in idc ( supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4503840 ) . characterizing the genomic context of the predicted circrnas
in dcis the 80% ( 443/552 ) of circular rnas belonged to the  exonic circrnas  class derived from back - spliced and the 20% belonged to cirnas ( circular intronic rna ) .
cirnas were a form of intron lariats that have been circularized at the branchpoint 25 linkage ; normally degraded from the 3 end up to the branchpoint , cirnas were stabilized in some way not yet clearly described .
idc showed a different profile : 55% ( 112/204 ) were cirnas , the remaining were 
the majority of the circular rnas identified had a length under the 12500 nucleotides in both the classes studied ( figures 1(a ) and 1(b ) and supplementary figure 1 ) .
the circrnas production can involve more than one exon , we found up to 16 exons involved both in dcis and idc . as expected
, there was a significant positive spearman 's rank correlation between the length and the number of exons involved in the circularization in dcis ( rho = 0.844 , 1 tail p value < 2.2e  16 ) and idc too ( rho = 0.630 , 1 tail p value < 2.2e  16 ) .
the chromosomes ( chr ) most represented in terms of circrnas were showed in figures 1(c ) and 1(d ) : chr1 , chr2 , and chr3 in dcis , while in idc there were chr2 , chr3 , chr10 , and chr19 .
two circular rnas , belonging to two different genes , fbxw4 and psma7 , were found in common between the idc samples and mcf7 cell line .
these data did not reflect the gene expression profiled studies and they may suggest that these discrepancies could be due to the different technology used , or that circrnas would be mostly related to the environment where the cancer cells were growing .
a panel of 18 circrnas were both expressed in at least one dcis and idc sample ( table 1 and supplementary figure 2 ) .
only one coding gene , sec62 ( also named tloc1 ) , showed two circular rnas that differed in terms of number of the exons involved .
these two circrnas were located in the chromosome region 3q26.2 , that it is interestingly frequent amplified in several cancers .
the longest in terms of nucleotides ( 11414  nts ) was the hsa - circ-0001358 ( alias hsa - circ-001803 ) , while the second was hsa - circ-0122662 , 8920  nts long .
there were no significant differences in terms of expression between these two circrnas comparing the levels of expression of dcis and idc classes ( wilcoxon - mann - whitney , p value = 0.8 ) .
the sec62 mrna was expressed more than the circular rnas isoforms ( figure 2(b ) ) and the biological trend of expression suggested a positive correlation between the mrna and the two circular rnas ( figures 2(c ) and 2(d ) ) , but this hypothesis was not significantly confirmed by the spearman rank test . to figure out the possible involvements of these circrnas as
 sponges  for the mirnas , we investigated if there were possible target binding sites in their sequences .  
table 2 listed five possible mirnas that can interact with the hsa - circ-0001358 , no interactions were found for the hsa - circ-0122662 .
the mirnas interacting with the circrna were obtained using the starbase human pan cancer tool .
the alignment scores of each mirnas were showed in supplementary figure 3 . of note ,
a decreased expression of microrna-200 family ( in table 2 were listed three members : mir-200b-3p , mir-200c-3p , and mir-429 - 3p ) was associated to migration and invasion of breast cancer cells in vitro in several reports in literature [ 23 , 24 ] .
therefore , we investigated the most important genes involved in epithelial mesenchymal transitions ( emt ) and that were targeted by the microrna-200 family to figure out a putative circrna / mirna / mrna axis involved in dcis to idc progression ( supplementary table 2 ) .
no significant correlations were found investigating the hsa - circ-0001358 and the zeb1 ( zinc finger e - box binding homeobox 1 ) , zeb2 ( zinc finger e - box binding homeobox 2 ) , and vim ( vimentin ) mrnas levels .
intriguingly , two targets of the mirnas showed an opposite behavior : bmi-1 ( b lymphoma mo - mlv insertion region 1 homolog ) positively correlated ( rho = 0.886 and p values = 0.02 ) with the circ-0001358 , instead fn1 ( fibronectin 1 ) showed a negative correlation ( rho = 0.886 and p values = 0.02 ) .
lastly , using circ2traits , we searched the measure of likelihood of the hsa - circ-0001358 , to figure out possible association with the 174 disease reported in the circ2traits database .
breast cancer was the third on twenty - four significantly associated disease ( p value = 0.002 , supplementary table 3 ) .
interestingly , we found many circrnas expressed only in dcis or idc , that could represent a difference in terms of selected dynamic expression .
this data will be in depth investigated for a better understanding of the dcis progression to invasive .
we have described a panel of predicted circrnas investigating a pilot small cohort of samples having dcis and idc synchronous .
we have to take into account two limitations of this study : ( i ) since the majority of circrnas were expressed at very low levels , and we analyzed total rna sequencing data without the use of rnase r , an enzyme that digests linear rnas but preserves circrnas , the number of circrnas identified was lower respect the media of other reports in literature .
therefore only an rnase r treatment will permit to enrich circrnas and then to further verify the existence of the predicted circular rnas .
( ii ) the second weakness point was the small size of this cohort . to further address the potential effect of the predicted circrnas , it will be fundamental enlarge the cohort and perform molecular biological experiments that might permit to validate these new findings .
altogether these data try also to shed light on the possible involvement of a circrna / mirna / mrna axis , but these data are still preliminary and in need of a further validation . in the next future
, this class of noncoding transcripts , still largely uncharted , will be revealed important in biomedical studies , especially in those seeking to find useful marker for diagnosis and prognosis and maybe to develop new therapeutics targets .
 the recent advantage obtained by next generation sequencing allows a depth investigation of a new  old  kind of noncoding transcript , the circular rnas . 
 circular rnas are nontranslated rnas , typically nonpolyadenylated , with a resistance to exonucleases that gives them the ability to be more stable than the common linear rna isoforms . 
 we used a bioinformatic detection tool ( circexplorer ) to research predictive circrnas from the next generation sequenced data of five samples of ductal in situ carcinoma ( dcis ) and matched adjacent invasive ductal carcinoma ( idc ) . 
 furthermore , we also investigated the circular rnas expressed in mcf7 , an invasive breast ductal carcinoma cell line . 
 we described the genomic context of the predicted circular rnas and we address the hypothetical possible functional roles . 
 this study showed a perspective of a panel of predictive circrnas identified and the function that circrnas could exert . 
1. Introduction
2. Material and Methods
3. Results and Discussion
4. Conclusion