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HOXD-AS1 is a novel lncRNA encoded in HOXD cluster and a marker of neuroblastoma progression revealed via integrative analysis of noncoding transcriptome.

Yarmishyn AA, Batagov AO, Tan JZ, Sundaram GM, Sampath P, Kuznetsov VA, Kurochkin IV - BMC Genomics (2014)

Bottom Line: We found that these lncRNAs fall into three distinct classes according to their statistical distribution by length.Studying retinoid acid (RA) response of SH-SY5Y cell line, a model of human metastatic neuroblastoma, we found that HOXD-AS1 is a subject to morphogenic regulation, is activated by PI3K/Akt pathway and itself is involved in control of RA-induced cell differentiation.Knock-down experiments revealed that HOXD-AS1 controls expression levels of clinically significant protein-coding genes involved in angiogenesis and inflammation, the hallmarks of metastatic cancer.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Long noncoding RNAs (lncRNAs) constitute a major, but poorly characterized part of human transcriptome. Recent evidence indicates that many lncRNAs are involved in cancer and can be used as predictive and prognostic biomarkers. Significant fraction of lncRNAs is represented on widely used microarray platforms, however they have usually been ignored in cancer studies.

Results: We developed a computational pipeline to annotate lncRNAs on popular Affymetrix U133 microarrays, creating a resource allowing measurement of expression of 1581 lncRNAs. This resource can be utilized to interrogate existing microarray datasets for various lncRNA studies. We found that these lncRNAs fall into three distinct classes according to their statistical distribution by length. Remarkably, these three classes of lncRNAs were co-localized with protein coding genes exhibiting distinct gene ontology groups. This annotation was applied to microarray analysis which identified a 159 lncRNA signature that discriminates between localized and metastatic stages of neuroblastoma. Analysis of an independent patient cohort revealed that this signature differentiates also relapsing from non-relapsing primary tumors. This is the first example of the signature developed via the analysis of expression of lncRNAs solely. One of these lncRNAs, termed HOXD-AS1, is encoded in HOXD cluster. HOXD-AS1 is evolutionary conserved among hominids and has all bona fide features of a gene. Studying retinoid acid (RA) response of SH-SY5Y cell line, a model of human metastatic neuroblastoma, we found that HOXD-AS1 is a subject to morphogenic regulation, is activated by PI3K/Akt pathway and itself is involved in control of RA-induced cell differentiation. Knock-down experiments revealed that HOXD-AS1 controls expression levels of clinically significant protein-coding genes involved in angiogenesis and inflammation, the hallmarks of metastatic cancer.

Conclusions: Our findings greatly extend the number of noncoding RNAs functionally implicated in tumor development and patient treatment and highlight their role as potential prognostic biomarkers of neuroblastomas.

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Related in: MedlinePlus

Pipeline for the automated annotation of Affymetrix U133 lncRNAs. Blocks represent data sets emerging from the analysis and rules used for their generation. Numbers represent the number of entries in each dataset.
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Figure 1: Pipeline for the automated annotation of Affymetrix U133 lncRNAs. Blocks represent data sets emerging from the analysis and rules used for their generation. Numbers represent the number of entries in each dataset.

Mentions: The probe sets of Affymetrix U133 chips were designed to detect not only transcripts of protein-coding genes, but also many transcripts currently annotated as ESTs and non-annotated RNAs(30), some of them can measure expression of unknown lncRNAs. Our goal was to find such probe sets. The 54,675 probe sets of Affymetrix microarrays U133A, U133B and U133Plus-2.0 were subjected to the filtration by quality and uniqueness of transcript matching according to the criteria of APMA database described by us earlier [30] (Figure 1). Following the database annotation, 7,587 probe sets matching unique genomic locations of RNAs not annotated as protein mRNAs were selected. Probe sets overlapping with exons of any known proteins (RefSeq) on the same DNA strand were excluded from the analysis. Thus, 1,586 probe sets were selected (Additional file 1), each of them matched a single transcript. To ensure that the selected transcripts do not encode proteins, we analyzed them with CRITICA software that identifies protein-coding sequences by potential presence of codons in the transcript sequences [31,32]. All 1,586 transcripts passed this filter and, therefore, they were considered as probe sets potentially measuring expression of lncRNA genes. To find which of these genes can be considered as true lncRNA candidates, their length distribution was analyzed. To ensure the reliability of the results, the sequences of individual Affymetrix probe sets were independently scanned against the 23,898 sequences of lncRNA transcripts annotated by Gencode consortium (v.19) using NCBI BLAST tool. Only 4,572 transcripts were found to have uniquely matching probe sets, 549 of which were present among our 1,586 ncRNA candidates. Since there was a limited overlap between the candidates and the Gencode-annotated lncRNAs, we considered the 549 lncRNAs as a high-confidence subset embedded in our data.


HOXD-AS1 is a novel lncRNA encoded in HOXD cluster and a marker of neuroblastoma progression revealed via integrative analysis of noncoding transcriptome.

Yarmishyn AA, Batagov AO, Tan JZ, Sundaram GM, Sampath P, Kuznetsov VA, Kurochkin IV - BMC Genomics (2014)

Pipeline for the automated annotation of Affymetrix U133 lncRNAs. Blocks represent data sets emerging from the analysis and rules used for their generation. Numbers represent the number of entries in each dataset.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4290621&req=5

Figure 1: Pipeline for the automated annotation of Affymetrix U133 lncRNAs. Blocks represent data sets emerging from the analysis and rules used for their generation. Numbers represent the number of entries in each dataset.
Mentions: The probe sets of Affymetrix U133 chips were designed to detect not only transcripts of protein-coding genes, but also many transcripts currently annotated as ESTs and non-annotated RNAs(30), some of them can measure expression of unknown lncRNAs. Our goal was to find such probe sets. The 54,675 probe sets of Affymetrix microarrays U133A, U133B and U133Plus-2.0 were subjected to the filtration by quality and uniqueness of transcript matching according to the criteria of APMA database described by us earlier [30] (Figure 1). Following the database annotation, 7,587 probe sets matching unique genomic locations of RNAs not annotated as protein mRNAs were selected. Probe sets overlapping with exons of any known proteins (RefSeq) on the same DNA strand were excluded from the analysis. Thus, 1,586 probe sets were selected (Additional file 1), each of them matched a single transcript. To ensure that the selected transcripts do not encode proteins, we analyzed them with CRITICA software that identifies protein-coding sequences by potential presence of codons in the transcript sequences [31,32]. All 1,586 transcripts passed this filter and, therefore, they were considered as probe sets potentially measuring expression of lncRNA genes. To find which of these genes can be considered as true lncRNA candidates, their length distribution was analyzed. To ensure the reliability of the results, the sequences of individual Affymetrix probe sets were independently scanned against the 23,898 sequences of lncRNA transcripts annotated by Gencode consortium (v.19) using NCBI BLAST tool. Only 4,572 transcripts were found to have uniquely matching probe sets, 549 of which were present among our 1,586 ncRNA candidates. Since there was a limited overlap between the candidates and the Gencode-annotated lncRNAs, we considered the 549 lncRNAs as a high-confidence subset embedded in our data.

Bottom Line: We found that these lncRNAs fall into three distinct classes according to their statistical distribution by length.Studying retinoid acid (RA) response of SH-SY5Y cell line, a model of human metastatic neuroblastoma, we found that HOXD-AS1 is a subject to morphogenic regulation, is activated by PI3K/Akt pathway and itself is involved in control of RA-induced cell differentiation.Knock-down experiments revealed that HOXD-AS1 controls expression levels of clinically significant protein-coding genes involved in angiogenesis and inflammation, the hallmarks of metastatic cancer.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Long noncoding RNAs (lncRNAs) constitute a major, but poorly characterized part of human transcriptome. Recent evidence indicates that many lncRNAs are involved in cancer and can be used as predictive and prognostic biomarkers. Significant fraction of lncRNAs is represented on widely used microarray platforms, however they have usually been ignored in cancer studies.

Results: We developed a computational pipeline to annotate lncRNAs on popular Affymetrix U133 microarrays, creating a resource allowing measurement of expression of 1581 lncRNAs. This resource can be utilized to interrogate existing microarray datasets for various lncRNA studies. We found that these lncRNAs fall into three distinct classes according to their statistical distribution by length. Remarkably, these three classes of lncRNAs were co-localized with protein coding genes exhibiting distinct gene ontology groups. This annotation was applied to microarray analysis which identified a 159 lncRNA signature that discriminates between localized and metastatic stages of neuroblastoma. Analysis of an independent patient cohort revealed that this signature differentiates also relapsing from non-relapsing primary tumors. This is the first example of the signature developed via the analysis of expression of lncRNAs solely. One of these lncRNAs, termed HOXD-AS1, is encoded in HOXD cluster. HOXD-AS1 is evolutionary conserved among hominids and has all bona fide features of a gene. Studying retinoid acid (RA) response of SH-SY5Y cell line, a model of human metastatic neuroblastoma, we found that HOXD-AS1 is a subject to morphogenic regulation, is activated by PI3K/Akt pathway and itself is involved in control of RA-induced cell differentiation. Knock-down experiments revealed that HOXD-AS1 controls expression levels of clinically significant protein-coding genes involved in angiogenesis and inflammation, the hallmarks of metastatic cancer.

Conclusions: Our findings greatly extend the number of noncoding RNAs functionally implicated in tumor development and patient treatment and highlight their role as potential prognostic biomarkers of neuroblastomas.

Show MeSH
Related in: MedlinePlus