BACKGROUND
neuroblastoma is the second commonest solid cancer in young children accounting for 9% of all childhood cancers.
it is characterised by a heterogeneous clinical behaviour ranging from spontaneous regression in 10% of all cases to rapid progression with unfavourable prognosis  <cit> .
amplified mycn leading to high mycn mrna and protein levels plays an important role in nb biology and is used as a powerful prognostic marker in nb risk stratification.
in addition, several other genetic abnormalities, including gain of 17q, 11q and 1p deletion have been associated with an aggressive nb phenotype  <cit> .
further, microarray technology has been used to study gene expression profiles in primary nbs.
patterns of differentially expressed genes among different nb subtypes as well as gene expression classifiers have emerged allowing a better prediction of patient's outcome than established risk markers  <cit> .
mirnas that regulate gene expression at the posttranscriptional level have been described to play a role in carcinogenesis via executing oncogenic or tumour suppressive functions  <cit> .
for example, let- <dig> mirnas are down-regulated in lung cancer and are known to target proto-oncogene ras transcripts  <cit> .
mir-17- <dig> cistron is to be overexpressed in human lung cancer and upregulated by c-myc  <cit> .
mir- <dig> and mir- <dig> are frequently deleted and down-regulated in chronic lymphocytic leukemias  <cit> .
the expression profiles of a small set of mirnas could be used to classify different types of cancer  <cit> .
more recently, profiling of a subset of known mirnas in neuroblastoma specimens suggested that mycn acts as a regulator of mirnas  <cit> .
however, the full repertoire of mirnas expressed in different cancer types, including neuroblastoma, is not yet available.
prognosis-relevant mirnas with putative oncogenic functions could have been missed in previous cloning efforts due to their silent state or low-expression.
there is evidence to date that neural lineage cells are particularly rich in mirna diversity  <cit> .
in this study, we aimed at cloning novel mirnas from nb cell lines and nb specimens that may have a role in nb biology.
here, we present the analysis of small rna libraries derived from neuroblastoma tumour specimens and cell lines and suggest several novel mirnas.
some of these mirnas reside within loci of cancer-associated rearrangements and are of special interest for future studies.
RESULTS
mirna cloning and abundance of previously annotated mirnas
for nb mirna library construction, we used rna derived from  <dig> tumour samples as well as two neuroblastoma cell lines, sh-ep  and kelly .
we followed the guidelines of lau et al.
ensuring that only those rna species were cloned which contained 5'-phosphate  <cit> .
the following cohort of tumours was used for cloning: four mycn amplified, three stage 4s mycn single-copy, two stage  <dig> mycn single-copy and four stage  <dig> mycn single-copy tumours .
* nb stage was evaluated according to inss criteria
approximately  <dig> clones per specimen or cell line were sequenced.
after linker unmasking,  <dig> small rna cdna sequences were subjected to further analyses.
the percentage of "not-known-mirna" sequences varied from 18% to 60% which corresponds to previously described fractions of other non-coding rnas, redundant sequences as well as novel mirnas in small rna libraries  <cit> .
our cloning project covered 27%  of mirnas currently present in the public mirna registry   <cit> .
<dig> mirnas were represented by only one read.
on the other hand, mir-124a  and -125b  yielded  <dig> and  <dig> reads, respectively .
mirna cloning was established as an independent semi-quantitative method to evaluate mirna abundance  <cit> .
this allowed us to conclude that mir-124a and -125b were the most abundant ones.
both mirnas are considered as having neuronal specificity  <cit> .
among the most frequently identified mirnas was mir- <dig> , a transcript with putative anti-apoptotic and tumour promoting activities  <cit> .
a difference in the abundance of certain mirnas was observed in mycn-amplified and non-amplified specimens.
in mycn-amplified tumours/cell line, we observed a higher abundance of mirnas from the mir-17/ <dig> cluster  that is known to be up-regulated by c-myc.
the average numbers for hsa-mir-17-5p and hsa-mir-20a reads in mycn-amplified and single-copy specimens were  <dig> versus  <dig>  and  <dig>  versus  <dig> , respectively.
a group of mirna sequences that clustered on chromosome  <dig> was exclusively found in the library of the mycn amplified tumour, mycnamp_nb <dig>  this argues for a cistronic manner of transcription of this group of mirna sequences.
beside annotated human mirnas we have cloned  <dig> sequences that have been originally identified in rat and mouse .
identification and validation of new mirnas
after removing redundant sequences and the sequences derived from other non-coding rnas, the remaining pool was checked in silico for the detection of the putative mirna precursors using mirpredict .
this analysis yielded  <dig> distinct sequences  which were classified as mirnas.
of these  <dig> mirnas,  <dig> sequences were mirna* forms of previously annotated mirna precursors .
hsa-mir- <dig> was cloned by the velculescu group, but then excluded from mirbase due to overlap with a trna gene  <cit> .
cloning of the 594* species described here provides evidence for the mirna nature of these sequences.
notes give the information which hairpin arm expresses the mirna* form.
other  <dig> sequences represent novel small rnas which have not been annotated.
two novel sequences were found adjacent to chromosomal localisations of known mirnas: mycnsc_nb5_ <dig> within a mirna cluster on chromosome  <dig> as well as mycnamp_nb2_ <dig> within a mirna cluster on chromosome  <dig> .
phylogenetic conservation was determined for each putative mirna and its surrounding with respect to chimpanzee, macaque, opossum, mouse, rat, dog, bull and chicken genomes .
we found that the cloned sequences with the exception of mycnamp_nb4_ <dig> are conserved either within all chosen species or primates.
the non-conserved mycnamp_nb4_ <dig> sequence seems to be the result of a short duplication within an intron of the dpp <dig> transcript .
however, homologous regions in chimpanzee and macaque genomes are also able to form hairpins which might give rise to mirnas.
novel mirnas do not share any homology with each other and therefore do not comprise a family.
searching against mirbase revealed that none of the new mirnas are related to the annotated mirnas in mirbase, except of one sequence, mycnamp_nb2_ <dig>  that is homologous to hsa-mir- <dig> and - <dig>  analysis of genomic locations of the individual mirnas showed that  <dig> of them are localized in extragenic regions.
<dig> sequences are found within introns of coding transcripts.
mycnsc_nb5_ <dig>  classified as a mirna by mirpredict, is found within a predicted u <dig> gene which reduces reliability of this sequence as a mirna .
the likely precursors of the novel mirnas  may be subdivided into two subgroups: structures with typical hairpin or borderline precursors.
the latter subgroup has features divergent from a canonical mirna hairpin, such as bulges , short stem  or "oscillating" hairpin , where the candidate mirna can reside on the 5' or 3'-arm.
lui et al.
reported the cloning of a subset of non-canonical mirnas, however the relevance of such rnas to the classical mirna pathway remains to be determined  <cit> .
notes include the information about genomic locations and additional sequence information.
based on the data listed above, we subjected  <dig> new mirnas to northern blot validation using biotinylated probes.
borderline case mycnsc_nb5_ <dig> sequences were not further examined.
the expression of each putative mirna was checked in a panel of human tissues, including different tumour specimens, normal brain, adrenal gland, spleen and skeletal muscle tissues.
<dig> of  <dig> cloned mirna* sequences and  <dig> sequences from individual hairpins were northern blot positive .
the other  <dig> sequences remained undetectable .
the probe for mycnsc_nb5_ <dig> yielded a signal around  <dig> nt which is higher than the estimated sequence size.
the probes for contig_chr_ <dig>  mycnsc_nb5_ <dig> and mycnamp_nb2_ <dig> yielded several bands different in length by 1– <dig> nucleotides.
some known mirnas show such patterns in northern blot analyses and intermediate products of mirna maturation have been observed, but there is no comprehensive explanation for these results  <cit> .
in normal tissues, northern blot-positive mirnas are preferentially expressed in brain, skeletal muscle tissue and adrenal gland, being less frequently expressed in spleen.
the detection of mirnas in skeletal muscle might be due to the presence of motor neurons.
several mirnas showed differential expression in normal versus tumour specimens: contig_chr_ <dig>  kelly_ <dig>  mycnamp_nb2_ <dig> were expressed in tumours, but not in normal tissues.
on the other hand, we did not find mycnamp_nb2_ <dig> mirna in tumour specimens, but we found strong expression of this sequence in brain.
CONCLUSIONS
recently, several studies have been performed aiming at directional cloning of tissue/tumour specific mirnas  <cit> .
the importance of such an approach is highlighted by the fact that patterns of novel mirnas from tumours of different lineages hardly overlap and may include mirnas with a functional role in tumourigenesis.
our study provides evidence for  <dig> new human mirnas cloned from neuroblastoma.
<dig> of them were originally cloned from other species and have not been annotated in the human mirna database.
<dig> sequences represent mirna* species of previously annotated sequences.
<dig> sequences, which have not been annotated before, are suggested to be novel mirnas.
some of these mirnas are found within regions of chromosomal rearrangements associated with cancer.
for example, new mirnas on 9p <dig> residing within nb-related aberration region are of special interest for future studies  <cit> .
mirna profiling of these novel sequences will allow us to clarify their relevance in nb development, progression and regression.

 neuroblastoma is the second commonest solid cancer in young children accounting for 9% of all childhood cancers. it is characterised by a heterogeneous clinical behaviour ranging from spontaneous regression in 10% of all cases to rapid progression with unfavourable prognosis. amplified mycn leading to high mycn mrna and protein levels plays an important role in nb biology and is used as a powerful prognostic marker in nb risk stratification. in addition, several other genetic abnormalities, including gain of 17q, 11q and 1p deletion have been associated with an aggressive nb phenotype. further, microarray technology has been used to study gene expression profiles in primary nbs. patterns of differentially expressed genes among different nb subtypes as well as gene expression classifiers have emerged allowing a better prediction of patient's outcome than established risk markers. mirnas that regulate gene expression at the posttranscriptional level have been described to play a role in carcinogenesis via executing oncogenic or tumour suppressive functions. for example, let- mirnas are down-regulated in lung cancer and are known to target proto-oncogene ras transcripts. mir-17- cistron is to be overexpressed in human lung cancer and upregulated by c-myc. mir- and mir- are frequently deleted and down-regulated in chronic lymphocytic leukemias. the expression profiles of a small set of mirnas could be used to classify different types of cancer. more recently, profiling of a subset of known mirnas in neuroblastoma specimens suggested that mycn acts as a regulator of mirnas. however, the full repertoire of mirnas expressed in different cancer types, including neuroblastoma, is not yet available. prognosis-relevant mirnas with putative oncogenic functions could have been missed in previous cloning efforts due to their silent state or low-expression. there is evidence to date that neural lineage cells are particularly rich in mirna diversity. in this study, we aimed at cloning novel mirnas from nb cell lines and nb specimens that may have a role in nb biology. here, we present the analysis of small rna libraries derived from neuroblastoma tumour specimens and cell lines and suggest several novel mirnas. some of these mirnas reside within loci of cancer-associated rearrangements and are of special interest for future studies.  mirna cloning and abundance of previously annotated mirnas for nb mirna library construction, we used rna derived from tumour samples as well as two neuroblastoma cell lines, sh-ep and kelly. we followed the guidelines of lau et al. ensuring that only those rna species were cloned which contained 5'-phosphate. the following cohort of tumours was used for cloning: four mycn amplified, three stage 4s mycn single-copy, two stage mycn single-copy and four stage mycn single-copy tumours. * nb stage was evaluated according to inss criteria approximately clones per specimen or cell line were sequenced. after linker unmasking, small rna cdna sequences were subjected to further analyses. the percentage of "not-known-mirna" sequences varied from 18% to 60% which corresponds to previously described fractions of other non-coding rnas, redundant sequences as well as novel mirnas in small rna libraries. our cloning project covered 27% of mirnas currently present in the public mirna registry. mirnas were represented by only one read. on the other hand, mir-124a and -125b yielded and reads, respectively. mirna cloning was established as an independent semi-quantitative method to evaluate mirna abundance. this allowed us to conclude that mir-124a and -125b were the most abundant ones. both mirnas are considered as having neuronal specificity. among the most frequently identified mirnas was mir-, a transcript with putative anti-apoptotic and tumour promoting activities. a difference in the abundance of certain mirnas was observed in mycn-amplified and non-amplified specimens. in mycn-amplified tumours/cell line, we observed a higher abundance of mirnas from the mir-17/ cluster that is known to be up-regulated by c-myc. the average numbers for hsa-mir-17-5p and hsa-mir-20a reads in mycn-amplified and single-copy specimens were versus and versus, respectively. a group of mirna sequences that clustered on chromosome was exclusively found in the library of the mycn amplified tumour, mycnamp_nb this argues for a cistronic manner of transcription of this group of mirna sequences. beside annotated human mirnas we have cloned sequences that have been originally identified in rat and mouse. identification and validation of new mirnas after removing redundant sequences and the sequences derived from other non-coding rnas, the remaining pool was checked in silico for the detection of the putative mirna precursors using mirpredict. this analysis yielded distinct sequences which were classified as mirnas. of these mirnas, sequences were mirna* forms of previously annotated mirna precursors. hsa-mir- was cloned by the velculescu group, but then excluded from mirbase due to overlap with a trna gene. cloning of the 594* species described here provides evidence for the mirna nature of these sequences. notes give the information which hairpin arm expresses the mirna* form. other sequences represent novel small rnas which have not been annotated. two novel sequences were found adjacent to chromosomal localisations of known mirnas: mycnsc_nb5_ within a mirna cluster on chromosome as well as mycnamp_nb2_ within a mirna cluster on chromosome. phylogenetic conservation was determined for each putative mirna and its surrounding with respect to chimpanzee, macaque, opossum, mouse, rat, dog, bull and chicken genomes. we found that the cloned sequences with the exception of mycnamp_nb4_ are conserved either within all chosen species or primates. the non-conserved mycnamp_nb4_ sequence seems to be the result of a short duplication within an intron of the dpp transcript. however, homologous regions in chimpanzee and macaque genomes are also able to form hairpins which might give rise to mirnas. novel mirnas do not share any homology with each other and therefore do not comprise a family. searching against mirbase revealed that none of the new mirnas are related to the annotated mirnas in mirbase, except of one sequence, mycnamp_nb2_ that is homologous to hsa-mir- and - analysis of genomic locations of the individual mirnas showed that of them are localized in extragenic regions. sequences are found within introns of coding transcripts. mycnsc_nb5_ classified as a mirna by mirpredict, is found within a predicted u gene which reduces reliability of this sequence as a mirna. the likely precursors of the novel mirnas may be subdivided into two subgroups: structures with typical hairpin or borderline precursors. the latter subgroup has features divergent from a canonical mirna hairpin, such as bulges, short stem or "oscillating" hairpin, where the candidate mirna can reside on the 5' or 3'-arm. lui et al. reported the cloning of a subset of non-canonical mirnas, however the relevance of such rnas to the classical mirna pathway remains to be determined. notes include the information about genomic locations and additional sequence information. based on the data listed above, we subjected new mirnas to northern blot validation using biotinylated probes. borderline case mycnsc_nb5_ sequences were not further examined. the expression of each putative mirna was checked in a panel of human tissues, including different tumour specimens, normal brain, adrenal gland, spleen and skeletal muscle tissues. of cloned mirna* sequences and sequences from individual hairpins were northern blot positive. the other sequences remained undetectable. the probe for mycnsc_nb5_ yielded a signal around nt which is higher than the estimated sequence size. the probes for contig_chr_ mycnsc_nb5_ and mycnamp_nb2_ yielded several bands different in length by 1– nucleotides. some known mirnas show such patterns in northern blot analyses and intermediate products of mirna maturation have been observed, but there is no comprehensive explanation for these results. in normal tissues, northern blot-positive mirnas are preferentially expressed in brain, skeletal muscle tissue and adrenal gland, being less frequently expressed in spleen. the detection of mirnas in skeletal muscle might be due to the presence of motor neurons. several mirnas showed differential expression in normal versus tumour specimens: contig_chr_ kelly_ mycnamp_nb2_ were expressed in tumours, but not in normal tissues. on the other hand, we did not find mycnamp_nb2_ mirna in tumour specimens, but we found strong expression of this sequence in brain.  recently, several studies have been performed aiming at directional cloning of tissue/tumour specific mirnas. the importance of such an approach is highlighted by the fact that patterns of novel mirnas from tumours of different lineages hardly overlap and may include mirnas with a functional role in tumourigenesis. our study provides evidence for new human mirnas cloned from neuroblastoma. of them were originally cloned from other species and have not been annotated in the human mirna database. sequences represent mirna* species of previously annotated sequences. sequences, which have not been annotated before, are suggested to be novel mirnas. some of these mirnas are found within regions of chromosomal rearrangements associated with cancer. for example, new mirnas on 9p residing within nb-related aberration region are of special interest for future studies. mirna profiling of these novel sequences will allow us to clarify their relevance in nb development, progression and regression. 
 reads corresponding to individual sequences were classified as novel mirnas, including mirna* species of known mirnas. neuroblastoma is an embryonal tumour consisting of neural crest-derived undifferentiated cells and is characterised by variable clinical courses ranging from spontaneous regression to therapy-resistant progression. however, the full repertoire of mirnas expressed in nbs is not available. recent advances identified a subset of mirnas with putative function in nb biology. some of these newly identified sequences reside within frequently altered chromosomal regions in nb tumours and may play a role in nb biology.
 micrornas are a novel class of gene expression regulators implicated in cancer biology. neuroblastoma is an embryonal tumour consisting of neural crest-derived undifferentiated cells and is characterised by variable clinical courses ranging from spontaneous regression to therapy-resistant progression. recent advances identified a subset of mirnas with putative function in nb biology. however, the full repertoire of mirnas expressed in nbs is not available.  we describe mirna profiles of nb specimens and nb cell lines as determined by mirna cloning. a total of sequences were sequenced and analysed by a mirna prediction tool. our library covered 27% mirnas known to date. reads corresponding to individual sequences were classified as novel mirnas, including mirna* species of known mirnas. expression of new mirna* forms and individual sequences was supported by northern blotting. most of the novel mirna genes are not related to each other and do not share homology with the annotated sequences in the public mirna database, but they are conserved within mammals or have close homologues in primates genomes.  we provide evidence for new mirna and mirna-like sequences. some of these newly identified sequences reside within frequently altered chromosomal regions in nb tumours and may play a role in nb biology. 