review article long noncoding rnas as new...

Review ArticleLong Noncoding RNAs as New Architects in Cancer EpigeneticsPrognostic Biomarkers and Potential Therapeutic Targets

Didier Meseure1 Kinan Drak Alsibai1 Andre Nicolas1

Ivan Bieche2 and Antonin Morillon3

1Platform of Investigative Pathology Institute Curie 75248 Paris France2Department of Pharmacogenomics Institute Curie and UMR 745 INSERM University Descartes 75248 Paris France3UMR 3244 Non-Coding RNA Epigenetics and Genome Fluidity Institute Curie 75248 Paris France

Correspondence should be addressed to Didier Meseure didiermeseurecuriefr

Received 25 October 2014 Accepted 6 May 2015

Academic Editor Konstantinos Arnaoutakis

Copyright copy 2015 Didier Meseure et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Recent advances in genome-wide analysis have revealed that 66 of the genome is actively transcribed into noncoding RNAs(ncRNAs) while less than 2 of the sequences encode proteins Among ncRNAs high-resolution microarray and massivelyparallel sequencing technologies have identified long ncRNAs (gt200 nucleotides) that lack coding protein function LncRNAsabundance nuclear location and diversity allow them to create in association with protein interactome a complex regulatorynetwork orchestrating cellular phenotypic plasticity via modulation of all levels of protein-coding gene expression WhereaslncRNAs biological functions and mechanisms of action are still not fully understood accumulating data suggest that lncRNAsderegulation is pivotal in cancer initiation and progression andmetastatic spread through variousmechanisms including epigeneticeffectors alternative splicing and microRNA-like molecules Mounting data suggest that several lncRNAs expression profiles inmalignant tumors are associated with prognosis and they can be detected in biological fluids In this review we will briefly discusscharacteristics and functions of lncRNAs their role in carcinogenesis and their potential usefulness as diagnosis and prognosisbiomarkers and novel therapeutic targets

1 Long Noncoding RNAs and FunctionalOrganization of the Genome

11 Genome and Noncoding RNA Large-scale genomic tech-nologies (high-resolution microarray whole genome andRNA sequencing) combined with bioinformatics analyseshave profoundly changed the genome organization under-standing Unexpectedly these global transcriptional analysesrevealed that 66 of the genome is transcribed and 80presents biochemical marks of active transcription whereasless than 2 encodes proteins [1] Discovery of a ldquosecondgenetic coderdquo consisting of ncRNAs has changed traditionalconcept of genomic organization characterized by presenceof genes encoding islets scattered in a sea of repeats and non-transcribed proteins Recently high-throughput techniqueshave identified 58648 human lncRNAs [2]

12 Classification of Noncoding RNAs NcRNAs are classi-fied into two categories according to their structural orregulatory properties and size This length arbitrarily setto 200 nucleotides (nt) corresponds to the threshold ofsensitivity of RNA extraction methods and can differentiatelncRNAs from short and medium ncRNAs such as microR-NAs (miRNAs) small nucleolar RNAs (snoRNAs) and piwiRNAs (piRNAs) [3] (Table 1) Although functions of mostncRNAs are currently still largely uncharacterized recentstudies have involved them in mechanisms implicated inimportant biological functions and in various pathologicconditions including neurodegenerative diseases and cancer[4 5] It is likely that development of new diagnostic andprognostic classifications involving ncRNAs refine medicalpractice and that ncRNAs may be useful potential targetsfor novel anticancer therapies In this review we propose to

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 320214 14 pageshttpdxdoiorg1011552015320214

2 BioMed Research International

Table 1 Type and main function of no-coding RNAs

Type Size (nt) FunctionsShort ncRNAs 19ndash31

miRNAs Targeting of mRNAs regulation of proliferation differentiation and apoptosisinvolved in human development

siRNAs Posttranscriptional gene silencing defense against pathogenic nucleic acidstiRNAs Regulation of transcription by targeting epigenetic silencing complexes

piRNAs Transposon repression DNA methylation development of germ cell stemself-renewal and retrotransposon silencing

tel-sRNAs Epigenetic regulationMid-size ncRNAs le200

snoRNAs rRNA modificationsPASRs Regulation of the transcription of protein-coding genesTSSa-RNAs Maintenance of transcriptionPROMPTs Activation of transcriptioncrasiRNAs Recruitment of heterochromatin andor centromeric proteins

Long ncRNAs gt200lincRNAs Involvement in biological processes such as dosage compensation andor imprintingIntronic lncRNAs Possible link with posttranscriptional gene silencing

T-UCRs Regulation of miRNA and mRNA levels and antisense inhibitors for protein-codinggenes or other ncRNAs

TERRAs Negative regulation of telomere length and activity through inhibition of telomerasePseudogene RNAs Regulation of tumor suppressors and oncogenes by acting as microRNA decoyslncRNAs with dual functions Modulate gene expression through diverse mechanisms

summarize recent literature data on lncRNAs their involve-ment in carcinogenesis and their value as biomarkers andpotential therapeutic targets

13 Definition of lncRNAs LncRNAs transcripts are definedby a length greater than 200 nts without any potential oftranslation [6] This definition is however arbitrary andcannot now be based on a set of physical biochemicalstructural or functional criteria Length is not an absolutecriterion because ncRNAs smaller than 200 nts (BC1 snaR)are included in the lncRNAs subgroup There is no specifictranscription of lncRNAs the transcriptional machinerybeing common to lncRNAs and mRNAs Lack of readingframe cannot also be a discriminating factor betweenmRNAsand ncRNAs because 50 of lincRNAs contain regions withhigh translational potential comparable to those of mRNAssuggesting that they are actively exported to cytoplasm andtranslated into short polypeptides albeit certainly not activeNoncoding characteristic is then not specific to lncRNAs asbifunctional coding and noncoding lncRNAs and lncRNAscontaining other ncRNAs (miRNAs snoRNAs) have recentlybeen discovered Several mRNAs lose their ability to encodeproteins (Xist) while others acquire coding function (SRA)[7] Furthermore by incorporating multiple exons fromcoding and noncoding genes alternative splicing generatesambiguous transcripts beyond all classifications Neverthe-less this definition appears currently to be the best that couldbe used waiting for a functional characterization of lncRNAs

14 Investigative Methods and Annotation of lncRNAs Thefirst lncRNAs were discovered by using functional geneticapproaches based on their relation to specific cellular mecha-nisms (i) Ubxn transcriptional interference-induced repres-sion by trithorax-promoted lncRNAbdx (ii) sex chromosomedosage compensation and Xist-induced X chromosomeand (iii) inactivation and genomic imprinting promoted byantisense lncRNAs Airn and Kcnq1 These seminal studiesestablished the concept of functional lncRNAs acting astranscriptional regulators involved in important and diversebiological processes Over the past two decades systematicglobal sequencing of cDNA libraries yielded comprehensiveknowledge of the transcribed but noncoding component ofthe genome These transcriptomic analyses have identifiednumerous lncRNAs RNA immunoprecipitation and siRNA-induced inactivation techniques led to functional validationof many lncRNAs

Currently lncRNAs identification and functional charac-terization are based on an experimental approach combining(i) discovery of new noncoding transcripts using RNA-seqand ChIP-seq (ii) annotation of transcribed regions extentand quantity of products transcribed through bioinformaticanalysis (iii) lncRNAs quantification in various cellular andtissue types and conditions (iv) coexpression or coregulationstudies and (v) gain and loss functional tests on cell linesand xenografts (siRNAs ASO TALEN and CRISPRCAS9)Advantage of RNA-seq technique resides in its sensibilityallowing most weakly expressed lncRNAs to be identified

BioMed Research International 3

However due to their frequent very low expression andcomplex exonintron structures it can be difficult to identifydifferent transcripts produced from alncRNA gene It is thennecessary to use other techniques through epigenetic analyses(markers of promoter regions or entire gene) targeted tran-scripts of interest sequencing after capture on a DNA chip(capture-RNAseq) and 51015840 and 31015840 extremities identificationanalyses [8ndash11]

Systematic large-scale projects have contributed a lot tothe systematic characterization of mammalian lncRNAs Forinstance the Fantom (Functional Annotation of the Mam-malian genome) is an international project initiated by Japanin 2000 designed to identify and annotate entire transcripts ofmouse Results published identified 15000 lncRNAs in 200223000 in 2005 and 34000 in 2006 with a transcribed genomefraction of 63 The ENCODE project initiated in 2003 isdesigned to identify map and make public all functionalelements of the human genome This project led to a catalogof lncRNAswith 10000 genes producing 15000 lncRNAsVeryrecently 58648 lncRNAs were identified by using the TCGAdatabase of which 79 were previously unannotated [2]

15 Properties of lncRNAs LncRNAs genes outnumber thoseof short ncRNAs and are probably more abundant thanmRNA genes LncRNAs are very heterogeneous in sizewith some extending over tens of kilobases (kb) They aretranscribed in all regions of eukaryotic genome particularlyduring development and are remarkably specific for a givencellular and tissue type

LncRNAs genes share many biochemical characteristicswith proteins coding genes predominant action of RNApoly-merase II epigenetic profiles cotranscriptionalmodifications(51015840Cap pre-lncRNAs alternative splicing and 31015840 polyadeny-lation) and exons and introns size However when lncRNAsare compared to mRNAs they appear less stable and shorterwith fewer exons a less well defined reading frame andmanyrepeat sequences (LINE SINE) A minority group of poorlycharacterized lncRNAs is represented by nonpolyadenylatedlncRNAs synthesized by RNA polymerase III and lncRNAssynthesized via alternative splicing and snoRNAs

Depending on their relative position to the nearest codinggenes several categories of lncRNAs can be identified asfollows

(i) Intragenic Regions Intragenic IncRNAsmay be subdivideddepending on how they overlap protein-coding genes or theirorientation [45]

(a) Long intronic ncRNAs (linRNAs) constitute themajor component of ncRNAs transcriptome UnlikelincRNAs tens of thousands linRNAs have recentlybeen identified but only few have been analyzedfunctionally [46]

(b) Sense lncRNAs are transcribed from the sense strandof protein-coding genes containing exons They mayhave some overlap with coding genes or cover entiresequence from an intron If it has been shown thatmost have no protein-coding potential several sense

lncRNAs can function as both ncRNAs and protein-coding genes (SRA ENOD40)

(c) Antisense lncRNAs (NAT) represent 32 of lncRNAsin humans and are poorly defined They are tran-scribed from the antisense strand of protein-codinggenes coding They may also have overlapping withexonic or intronic regions or cover entire sequencefrom an intron NATs are observed in many speciesincluding animals plants yeasts prokaryotes andviruses but have no sequence or conserved structurethat could be indicative of a particular functiondespite several highly conserved lincRNAs [47]

(ii) Intergenic Regions Intergenic lncRNAs (lincRNAs) arelocated in unannotated genomic regions and represent thebest studied class of lncRNAs LincRNAs are functionallncRNAs characterized by stability K4H36 active domainof transcription and tissue-specific expression They act intrans and are primarily involved in epigenetic regulation ofprotein-coding genes expression and maintenance of stemcells pluripotency [48]

(iii) Enhancer ncRNAs (eRNAs)Two types of lncRNA (multi-exonic or lacking intronswith bidirectional transcription) arederived from enhancer sequences Enhancer sequences regu-late temporal and specific expression of genes via cis or transmechanisms The human genome contains approximatelyone million enhancer sequences most likely controlling the19000 genes coding for proteins LncRNAs derived fromenhancer sequences play a crucial role in the formation ofchromatin loops that allow stabilization and association ofenhancer sequences with the promoter regions that initiatetranscription of their target genes [49]

(iv) Promoters PALR (promoter associated long ncRNA)

(v) Telomeric Regions TERRA (telomeric repeat containingRNAs)

Within cells lncRNAs are located in the nucleus accord-ing to their main protein-coding genes epigenetic regu-latory function A minority of lncRNAs are involved innucleocytoplasmic trafficking (NRON) mRNA stability andtranslational regulation Several lncRNAs (MALAT1) arecleaved byRNase P in a structural and regulatory intranuclearcomponent (speckle) and a small cytoplasmic tRNA-liketranscript LncRNAs topography recognition should help infinding new efficient lncRNAs-based targeted therapies [50]Finally lncRNAs can be precursors of ncRNAs (snoRNAmiRNA)

16 Modular Organization of the Genome and StructuralPlasticity of lncRNAs The genome has a modular architec-ture composed of complex transcriptional loci character-ized by close links between nucleotide sequences organizedin senseantisense and codingnoncoding transcripts Thus


over 50 of protein-coding genes are associated with com-plementary antisense noncoding transcripts cis-regulatingchromatin and adjacent genes expression although no sys-tematic study classified them carefully as lncRNAsMoreovercombined application of alternative splicing and transcrip-tional initiation and termination utilizes this modular archi-tecture to ensure transcriptional diversification illustratedby the very large number of lncRNAs isoforms [48] LncR-NAs structural plasticity and biochemical properties betterexplain the great diversity of their mechanisms of actionthan their mere nucleotide sequence Their organization insecondary and tertiary structures contributes to the creationof functional domains interacting with proteins (small lig-ands multiprotein complexes) or hybridizing with nucleicacids (mRNA miRNA and DNA) Their interactions lead inturn to lncRNAs allosteric conformational changes allowingthem to bind to other actors involved in gene expressionand mRNA translation Thus lncRNAs initiate regulatorynetworks with high complexity at epigenetic transcriptionaland posttranscriptional levels in order to transmit andcoordinate information flows in signaling pathways requiredfor eukaryotic cells functioning [51]

17 Conservation Evolution and Origin of lncRNAs LncR-NAs have been observed in many eukaryotes Converselyto protein-coding sequences lncRNAs have rapidly evolvedexplaining that lncRNAs orthologues to mammals are onlyfound in vertebrates Even in vertebrates DNA sequencesconservation is low and suggests that most lncRNAs are notfunctional due to insufficient selection pressure Nonethelessothers criteria could be taken into account in assessmentof their conservation including genomic localization tran-scriptional profile and tertiary structure

Current complexity of humanphysiology cannot be solelyexplained by expression of 20000 protein-coding genescomparable to that observed in Drosophila melanogaster andCaenorhabditis elegans but rather by parallel development ofa noncoding genome Unlike proteome amount of ncRNAshas increased during evolution and it seems to be a significantcorrelation between lncRNAs expression levels and complex-ity explaining that primates havemost lncRNAsThe numberof protein-coding genes cannot explain functioning of veryfinely regulated organs such as brain This additional level ofcomplexity observed in vertebrates may be partly related toexpression of lncRNAs with a high spatiotemporal specificityand their interactions with DNA mRNAs and proteinsLncRNAs could then be considered as molecular sensorsof environmental changes conferring evolutional plasticitythat contributes to development of life complexity Duringenvironmental changes lncRNAs located within intergenicsequences may serve as supports for new functions allowingbody to adapt new constraints Protein-coding genes couldgenerate transcripts from lncRNAs genes Conversely pro-teins may be synthesized from lncRNAs Recent data haveidentified 24 human protein-coding genes with noncodinghomologous genes in other species

Recent emergence of numerous lncRNAs suggests thatthey continue to be actively synthesized (i) from ancestralgenes that have lost their coding potential (ii) by genes or

other lncRNAs duplication transposition and mutation and(iii) de novo from intergenic DNA [52 53]

18 LncRNAs Functions and Mechanisms of Action Growingdata have demonstrated that lncRNAs exhibit the greatestdiversity among functional ncRNAs and play regulatory andstructural roles in embryogenesis stem cells pluripotencyallelic expression protein-coding genes regulation apoptosiscycle control growth differentiation and senescence Inpractice only a very limited number of lncRNAs (1) havebeen well characterized functionally in humans includingXist KCNQ10T1 AIR HotAir ANRIL HOTTIP MALAT1TERRA and HULC

LncRNAs play a role in stem cells and differentiationmaintenance of pluripotency and lineage differentiation inembryonic stem cells (ESCs) and induced pluripotent stemcells (iPSCs) Adult stem cells are finely regulated by lncRNAsnetworks that are targets of most of master pluripotencytranscription factors (Oct4 Sox2 Nanog cMyc and KLF4)[54]

LncRNAs are considered as crucial regulators coordinat-ing protein-coding genes expression by numerous mecha-nisms depending on their cellular localization and leading tomodifications of chromatin transcription and translation

These regulatory mechanisms are located at epigenetictranscriptional posttranscriptional and translational levelsas follows

(i) LncRNAs constitute a network of epigenetic modu-lators recruiting guiding and forming platforms that buildribonucleoprotein complexes at specific genomic sites 20ofhuman lncRNAs recruit multimolecular repressor activatoror chromatin remodeling complexes (PRC1PRC2 byANRILLSD1PRC2 by HOTAIR) whose subcatalytic units (EZH2EED BMI1 SUZ12 CBX7 CoREST and JARID1) interactby altering histone code and methylation profile Cis-actinglncRNAs adjacent to locus where they are transcribed actby transcriptional interference or by modifying chromatinTranscriptional interference allows inhibition of preinitial-ization complexes and interaction with transcription fac-tors Chromatin modifications result from recruitment ofcomplexes inhibiting (Polycomb by ANRIL) or activating(MLL by HOTTIP) gene expression Trans-lncRNAs operateindependently of complementarities sequences and act atdistance onmany genomic loci via specific DNAmotifsTheyinfluence genes expression by recruiting modifying chro-matin complexes binding transcriptional elongation factorsand inhibiting RNA polymerase [55]

(ii) LncRNAs associated with promoters (PaRNAs) andlncRNAs of enhancer type (eRNAs) can directly regulatetranscription of target genes by transcriptional activationor suppression PaRNAs function as protein coactivatorsfacilitating gene transcription PaRNAs of 50ndash200 nt areinvolved in repression of polycomb-targeted genes via mech-anisms allowing transcription of these lncRNAs from com-mon promoter regions to those of target genes regulatedby these noncoding transcripts Gene cis-repression is theresult of PRC2 complex (SUZ12) recruitment by PaRNAsattachment to promoter regions and modification of histonemethylation (H3K27me3) on target promoters ERNAs have


transcriptional activation functions of protein-coding genescoding including genes involved in embryonic developmentand differentiation (ncRNAa3 and TAL ncRNAa7 and SNAI1)[56ndash58]

(iii) LncRNAs can also act at posttranscriptional leveland are widely involved in biogenesis stability and transcrip-tional activity of mRNAs They regulate alternative splicingpromote trafficking direct cellular localization and promotemRNAs degradation LncRNAs also synthesize miRNAs andbuild sponge-like structures to prevent binding ofmiRNAs totheir targetmRNAs (CDR1-asciRS-7 circular RNA sponge formiR-7) [59]

(iv) LncRNAs can finally bind to inhibiting factors oftranslation interact with ribosomes and allow transport ofproteins

LncRNAs may participate in assembly of specializedintranuclear functional structures including speckles(MALAT1) paraspeckles (NEAT1) and polycomb body(TUG1)

Several lncRNAs particularly lincRNAs interact withfactors (Oct4 Sox2 Nanog c-Myc Klf4 Smad and Tcf3) andplay key roles in maintaining stem cells pluripotency

Moreover lncRNAs have four known molecular func-tions (Figure 1) (1) Signal lncRNAs regulate transcriptionalactivity or pathways (lincRNA-p21) (2) Guide lncRNAslink specific proteins belonging to chromatin remodelingcomplexes and recruit them to homology containing targetloci where genes silencing is promoted (HOTTIP XIST) (3)Decoy lncRNAs bind and titrate away proteins or RNAsIn the nucleus they can bind transcription factors or DNAmethyltransferase 1 (PANDA Gas5 MALAT1) whereas inthe cytoplasm they can function like a sponge to attract pro-teins andmiRNARISC complexes from theirmiRNA targets(4) Scaffold lncRNAs constitute adaptors that bindmolecularcomplexes and regulate gene expression (HOTAIR ANRILand TERCTERT) [18 60]

19 lncRNAs and Cancer Because lncRNAs are involved invarious and important physiological processes their dysfunc-tion should have important consequences for cell homeosta-sis Several recent studies have indeed shown that expressionof many lncRNAs varies significantly in different conditionscompared to healthy tissue Significance of these deregula-tions (consequence of a globally altered transcriptional statusor causative and driving abnormality) is still a matter ofdebate However transcripts of noncoding genome revealednew dimension of the molecular architecture of cancer andlncRNAs are involved in all stages of oncogenesis Geneexpression profiles analysis of various tumors showed thatlncRNAs are deregulated and functional studies have demon-strated that lncRNAs are implicated in general mechanismsof carcinogenesis Moreover genetic studies have revealedexistence of mutations in their primary sequences Sincemost genetic variants identified by genome-wide associationstudies (GWAS) are located outside coding genes many ofthese mutations may therefore affect lncRNAs

LncRNAs may regulate signaling pathways involved ininitiation tumor progression and metastatic spread similar

to protein-coding oncogenes and oncosuppressors PINCand PGEM1 were the first oncogenic lncRNAs found over-expressed in breast and prostate carcinomas Since thenmany other lncRNAs have been identified with oncogenicproperties (KRASP HULC HOTAIR MALAT1 HOTTIPANRIL and RICTOR) or oncosuppressive properties (MEG3GAS5 LincRNA-p21 PTENP1 TERRACCND1CyclinD1 andTUG1) Interestingly several lncRNAs may show both onco-genic and oncosuppressive activities depending on cellularcontext XIST noncoding transcript is overexpressed in maletumors and underexpressed in female tumors

(i) Oncogenic lncRNAs

(a) SRA (steroid receptor RNA activator) is a coactivatorfor steroid receptors and acts as an ncRNA foundin the nucleus and cytoplasm SRA regulates geneexpression mediated by steroid receptors throughcomplexing with proteins also containing steroidreceptor coactivator 1 (SRC-1) The SRA1 gene canalso encode a protein that acts as a coactivatorand corepressor SRA levels have been found to beupregulated in breast tumors where it is assumed thatincreased SRA levels change the steroid receptorsrsquoactions contributing to breast carcinogenesis Whileexpression of SRA in normal tissues is low it is highlyupregulated in breast uterus and ovary carcinomas[61ndash63]

(b) HOTAIR (HOX antisense intergenic RNA) is a longintergenic noncoding RNA with a length of 22 kb inHOXC locus and transcribed in antisense mannerHOTAIR regulates gene expression by modulatingchromatin structures It was the first lncRNA dis-covered to be involved in carcinogenesis Polycombgroup proteins mediate repression of transcriptionof thousands of genes that control differentiationpathways during development pluripotency and can-cer progression Target of PRC2 is HOXD locuson chromosome 2 where PRC2 in association withHOTAIR promotes transcriptional silencing ofmetas-tasis suppressor genes HOTAIR acts as a molecularscaffold remodeling chromatin by causing histonemodifications on target genes HOTAIR comprisestwo known chromatin modification complexes with51015840 region binding to PRC2 complex responsible forrepressive H3K27 methylation and 31015840 region bindingto LSD1 which initiates activating H3K4 demethyla-tion [64 65]

(c) ANRIL (antisense ncRNA in the INK4 locus) isa natural antisense transcript which activates thetwo polycomb repressor complexes PRC1 and PRC2resulting in chromatin reorganization with silencingof INK4b-ARF-INK4a This results in p15INK4bp14ARF and p16INK4a inhibition which are nor-mally implicated in cell cycle negative regulationsenescence and stress-induced apoptosis ANRILoverexpression in prostate carcinomas has shownsilencing of INK4b-ARF-INK4a and p15CDKN2B


Geneexpression

lncRNAs

(a) Signal lncRNAs affect expression of the downstream geneand regulate transcriptional activity or pathway

lncRNA

Protein

(b) Decoy lncRNAs titrate proteins away from chromatin orthey can function as decoy for miRNA target sites

lncRNA

(c) Guide lncRNAs can recruit chromatin modifying enzymesto target genes and function as guides

lncRNAs

(d) Scaffold lncRNAs can bring together multiple proteins toform ribonucleoprotein complexes

Figure 1 Molecular functions of lncRNAs

Repression mechanism is mediated by direct bindingto CBX 7 and SUZ12 2 members of PRC1 and PRC2respectively [66ndash68]

(d) MALAT1 (metastasis-associated lung adenocarci-noma transcript 1) is a lincRNA widely expressedin normal human tissues and overexpressed in avariety of breast colon liver pancreas prostateand uterus carcinomas MALAT1 locus at 11q131 hasbeen identified to harbor chromosomal translocationbreak points and mutations in breast and bladdercarcinomas It is localized in nuclear speckles andrecent studies have implicated MALAT1 in splicingEMT cell mobility ECM remodeling and metastaticspread [69]

(e) HULC (highly upregulated in liver cancer) has beensuggested to act as a ldquospongerdquo that inhibits miR-372by sequestering it away from potential mRNA targets[70]

(f) PCGEM1 (prostate cancer gene expression marker 1)is overexpressed in prostate cancer cells that promotestumor cells initiation and progression and protectsagainst chemotherapy-induced apoptosis Moreoverthe reciprocal negative control relationship betweenPCGEM1 and miR-145 regulates both prostate cancercells proliferation and tumor growth These resultsidentify PCGEM1 and associated regulators as possi-ble targets for prostate cancer therapy [71]

(ii) Tumor Suppressor lncRNAs

(a) MEG3 (maternally expressed gene 3) is a tran-script of the maternally imprinted gene normallyexpressed in pituitary cells MEG3 loss of expressionis observed in pituitary adenomas and meningiomasMEG3 acts by regulating p53 pathway P53 levelsare usually extremely low due to its rapid degrada-tion via ubiquitin-proteasome pathway P53 ubiqui-tination is mainly mediated by E3 ubiquitin ligaseMDM2 MEG3 downregulates MDM2 expressionincreases p53 protein level stimulates p53-dependenttranscription and enhances p53 binding to targetpromoters [72]

(b) GAS5 (growth arrest-specific 5) is widely expressedin embryonic and adult tissues GAS5 acts as astarvation or growth arrest-linked riborepressor forglucocorticoid receptors by inhibiting association ofthese receptors with their DNA recognition sequenceThis suppresses several responsive genes activationincluding gene encoding cellular inhibitor of apopto-sis 2 (cIAP2) GAS5 low expression levels have beenobserved in prostate and breast carcinomas [73 74]

(c) CCND1Cyclin D1 is transcribed from the promoterregion of the Cyclin D1 gene Cyclin D1 is a cell cycleregulator frequently mutated amplified and over-expressed in carcinomas CCND1Cyclin D1 recruitsthe RNA-binding protein TLS which undergoes


allosteric modification resulting in Cyclin D1 geneinhibition [75 76]

(d) LincRNA-p21 expression is directly induced by p53signaling pathway This lncRNA is implicated inglobal repression of genes interfering with p53 func-tion to regulate cellular apoptosis [77]

(e) TERRA (telomeric repeat containing RNA) isexpressed at chromosome ends TERRA upregulationupon experimentalmanipulation or in ICF (immuno-deficiency centromeric instability and facialanomalies) patients correlates with short telomeresTERRA transcription facilitates the 51015840-31015840 nucleaseactivity of Exo1 at chromosome ends providinga means to regulate the telomere shortening rateThereby telomere transcription can regulate cellularlifespan through modulation of chromosome endprocessing activities [78]

(iii) Oncogenic and Tumor Suppressor lncRNAs(a) H19 is expressed from the maternal allele and has

a pivotal role in genomic imprinting during cellgrowth and developmentThe locus containsH19 andIGF2 which are imprinted This leads to differentialexpression of both genes H19 from maternal andIGF2 from paternal alleleThis lncRNA presents bothoncogenic and suppressive properties although theexact mechanism is still elusive [79]

LncRNAs are implicated in cancer epigenetics Epigeneticsrefers to events that modulate activity of the genome withoutchanging its sequence It provides control of genome expres-sion and establishment of tissular and cellular-type specificgenes expression profiles The main epigenetic mechanismscomprise chemical modifications of DNA and histones (cyto-sinemethylation posttranslational modifications of histoneschromatin remodeling and nucleosome positioning) Thesechemical changes are reversible and controlled by enzymecomplexes directly connected to metabolic and signalingpathways as well as sensors of extra- and intracellularmicroenvironments Deregulation of these epigenetic mech-anisms has been demonstrated in cancer cells which have notranscriptional gene or protein expression while being free ofDNA damage Carcinogenesis is very frequently associatedwith abnormal signaling and epigenetic abnormalities mostof which are considered as significant oncogenic events(aberrant methylation of oncosuppressor genes) LncRNAsinteract with numerous proteins and in particular epigeneticregulators includingDNAmethyltransferases and enzymaticcomplexes modifying chromatin and nucleosomes [80]

(i) Methylation of DNA was first observed in cancerepigenetic alterations Cancer epigenome is characterized byglobal hypomethylation leading to genomic instability andhypermethylation of CpG islands which cause inactivationof genes oncosuppressors implicated in signaling pathways(DNA repair apoptosis and cycle cell regulation) or tran-scription factors involved in the control of these genes LncR-NAs bind to DNA methyltransferase guide it to promoter ofoncosuppressor genes and ensure transcriptional silencing

(ii) Nucleosomes can be destabilized and restructuredby remodeling complexes belonging to 4 families (SWISNFISWI CHD and INO80) Nucleosomes positioning andremodeling regulate gene expression by perturbating acces-sibility of DNA regulatory sequences by transcription factorsand transcription machinery Mutations and silencing ofsubunits of these complexes in various types of cancerhave been observed LncRNAs could reposition nucleosomesby interacting and guiding these remodeling complexes tospecific genomic regions where nucleosome restructuringwould ensure repression of oncosuppressive genes [80]

(iii) LncRNAs recruit histone modifying enzymes actingnear the transcription site of lncRNAs Histones modifyingenzymes read add or remove covalent links with change inaccessibility of chromatin and fixation of nonhistone proteineffectors that decode modified histone code Alterationsin the expression of histone-modifying enzymes (muta-tions overexpression) were observed in various carcinomasThese alterations inhibit oncosuppressive genes transcription(EZH2 histone methyltransferase responsible for repressiveH3K27me3 mark and silencing of p15 p16 and p19 histonedeacetylase responsible for loss of lysine acetylated H4) oractivate oncogenes transcription (demethylases and deacety-lases) [81]

Growing numbers of LncRNAs are implicated in numer-ousmechanisms characterizing hallmarks of cancerThey canact on proliferation via coactivation (SRA1) and inhibitionof elongation (RN7SK) LncRNAs allow escape mechanismsfrom suppressor pathways via competition (PFS) silencing(ANRIL) or ribonucleic repression (GAS5) Several lncRNAsare involved in replicative immortality by inhibiting telom-erase (TERRA) LncRNAs promote neoangiogenesis is byinhibiting HIF1A (AHIF MALAT1) They induce resistanceto cell death by acting on p53 and p21 (PCGEM1) andby reducing expression of proapoptotic genes (PANDA)Finally lncRNAs are involved in mechanisms of invasion andmetastasis via deregulation of alternative splicing (MALAT1)or trans-silencing of HOXD locus (HOTAIR)

2 LncRNAs as Biomarkers and TherapeuticTargets in Future Medical Practice

LncRNAs are emerging as integral functional components ofhuman genome and are now considered as critical regulatorsin molecular biology of cancer Recent data have demon-strated that lncRNAs are associated with cancer initiationtumor progression and metastatic spread Unlike mRNAsused as diagnostic and prognostic biomarkers while beingexpressed by numerous subtypes of malignant tumors lncR-NAs present cellular and tissular specificity and could serve asbiomarkers and therapeutic targets in cancer FurthermorelncRNAs secreted or released by apoptotic or necrotic tumorcells can be detected in blood plasma and urineThere seemsto be a significant correlation between levels of circulatingnucleic acids and genomic epigenetic or transcriptionalalterations associated with malignant tumors (Table 2)

(i) EsophagusThree lncRNA signatures have been identifiedrecently in oesophageal squamous cell carcinoma


Table 2 Representative lncRNAs involved in carcinogenesis and potential cancer biomarkers

Cancer type lncRNAs References

Esophagus ENST000004358851 XLOC 013014ENST000005479631 [12]

Stomach GCAT1 H19 SUMO1P3 [13ndash17]Colon and rectum HOTAIR uc73 [18 19]Liver HULC HOTAIR MALAT1 HOTTIP HEIH [20ndash22]Lung MALAT1 TUG1 BANCR GAS5 [23ndash28]Breast HOTAIR LincRNA-RoR UCA1 [29ndash32]Ovary HOTAIR [33]Bladder UCA1 H19 Linc-UBC1 MALAT1 [34 35]Prostate PCA3 PCAT1 PCGEM1 [36ndash39]Glioma H19 [40]Melanoma BANCR [41]

Oral cavity and nasopharynx HOTAIR lnc-C22orf32-1 lnc-AL3551491-1lnc-ZNF674-1 [4 42ndash44]

including ENST000004358851 XLOC 013014 andENST000005479631 The expression of these lncRNAsclassified the patients into two groups with significantlydifferent overall survival [12]

(ii) Stomach HOTAIR GCAT1 (gastric cancer-associatedtranscript 1) H19 and SUMO1P3 (small ubiquitin-like mod-ifier SUMO 1 pseudogene 3) are the main lncRNAS reportedas overexpressed in gastric carcinomas They are often asso-ciated with lymph node and distant metastasis suggestingthat they might serve as potential diagnostic and prognosticbiomarker [13ndash17] Moreover CCAT1 expression is closelyrelated to c-Myc activation [14] CCAT1 functions as anoncogene and may be used as biomarker and potentialtherapeutic target in gastric carcinoma [15 16]

(iii) Lower Digestive Tract HOTAIR overexpression isobserved in colorectal carcinomas with advanced stage andliver metastases [19] uc73 lncRNA is also associated withpoor overall survival in patient with colorectal carcinomas[18] These results place HOTAIR and uc73 lncRNA asreliable biomarkers for poor prognosis in colorectal cancer

(iv) Liver In hepatocellular carcinomas (HCC) HULC(highly upregulated in liver cancer) was the first lncRNAwith highly specific upregulation detected in blood [20]High plasma HULC rates were observed in patients withhigh grades HCC or with HBV+ status [21] Recent datahave shown that HBx could regulate HULC promoter toinduce HCC via oncosuppressor p18 silencing [22] HBxwas found to downregulate lncRNA-Dreh which can inhibithepatocellular growth and metastasis in vitro and in vivo

MALAT-1 and HOTAIR have been shown to be over-expressed in large cohorts of HCC patients Further-more HOTAIR is a prognostic biomarker for recurrenceafter liver transplantation SiRNA-mediated inhibition of

MALAT-1 and HOTAIR suppresses cancer cell viability andinvasion sensitizes TNF-120572 induces apoptosis and increaseschemotherapeutic sensitivity of HCC to cisplatin and dox-orubicin [82 83] HOTTIP (HOXA transcript at the distaltip) and HOXA13 were also found to be upregulated inHCC HOTTIP and HOXA13 levels were associated withHCC tumor progression metastasis and survival [84]HEIH(high expression in HCC) is another lncRNA overexpressedinHCCand an independent prognostic factor associatedwithrecurrence [85]

(v) Lung MALAT1 is a key prognostic biomarker formetastatic spread in lung adenocarcinomas [23] TUG1 (tau-rine upregulated gene 1) is generally downregulated in non-small cell lung carcinomas (NSCLC) In NSCLC patientsTUG1 low expression was associated with high TNM stagetumor size and poorer overall survival Univariate andmultivariate analyses revealed that TUG1 expression serves asan independent predictor for overall survival [24] HOTAIRwas initially reported to be highly expressed in NSCLCs withadvanced stage and lymph node metastasis [25] But morerecent meta-analyses study showed that it did not reach sta-tistical significance and thus it needs further investigations[26] In NSCLCs BANCR (BRAF activated noncoding RNA)expression is significantly decreased compared to normaltissues BANCR underexpression is considered as an inde-pendent prognostic factor and is associated with larger tumorsize advanced pathological stage metastasis distance andshorter overall survival Recently BANCR overexpressionwas found to play a key role in epithelial-mesenchymaltransition [27] In malignant pleural mesothelioma (MPM)GAS5 (growth arrest specific transcript 5) underexpressionwas observed compared to normal mesothelial tissue Con-verselyGAS5was upregulated upon growth arrest induced byinhibition of Hedgehog and PI3KmTOR signaling in MPMmodels [28]


(vi) Breast Breast cancer progression is correlated withHOTAIR activity in numerous recent studies The studycarried out by Chisholm and colleagues demonstratedHOTAIR overexpression in primary and metastatic breastcarcinoma tissues by using RNA in situ hybridization tech-nique [29] Another study focusing on HOTAIR revealed thedependence of HOTAIR expression on oestradiol produc-tion due to its promoter region via several estrogen responseelementsThis study also demonstrated thatHOTAIR knock-down induced apoptotic pathways in breast cancer celllines and suggested estrogen receptors as coregulators forHOTAIR expression [30] Conversely a recent study focusingon 348 primary breast carcinomas revealed that increasedDNA methylation led to HOTAIR downregulation and anunfavorable disease state questioning suitability of HOTAIRas negative prognostic biomarker in breast carcinomas [31]LincRNA-RoR suppresses p53 in response to DNA damagethrough interaction with heterogeneous nuclear ribonucleo-protein I (hnRNP I) Recent data demonstrated that hnRNPI can also form a functional ribonucleoprotein complex withUCA1 (urothelial carcinoma-associated 1) and increaseUCA1stability Of interest the phosphorylated form of hnRNP Ipredominantly located in the cytoplasm is responsible forthe interaction with UCA1 Although hnRNP I enhancesp27 translation through interaction with the 51015840-untranslatedregion of p27 mRNAs interaction of UCA1 with hnRNP Isuppresses p27 protein expression via competitive inhibitionIn support of this finding UCA1 seems to have an oncogenicrole in breast carcinogenesis both in vitro and in vivo Finallya negative correlation between p27 and UCA1 was foundTogether these results suggest an important role of UCA1 inbreast carcinogenesis [32]

(vii) Glioma H19miR-675 signaling was recently identifiedas a critical pathway in glioma progression By analyzinggene expression data H19 increased levels of expressionwere found in high grade glioma SiRNA-induced H19depletion inhibited invasion in glioma cells FurthermoreH19 expression was positively correlated with miR-675 andH19 inhibition reduced miR-675 expression Collectivelythese data suggest that H19 regulates glioma development byderiving miR-675 [40]

(viii) ENT TumorsRecentlyHOTAIRwas suggested to play apart in nasopharyngeal carcinoma (NPC)HOTAIR is impli-cated in NPC progression and patients with high HOTAIRlevels have poor clinical outcome with tumor recurrenceand distant metastasis [42 43] In primary NPCs upregu-lation of lnc-C22orf32-1 lnc-AL3551491-1 and lnc-ZNF674-1 has been also observed High levels of lnc-C22orf32-1 andlnc-ZNF674-1 are associated with advanced tumor stagesRecurrent NPC displayed a distinctive lncRNA expressionpattern with increased expression of lnc-BCL2L11-3 anddecreased expression of lnc-AL3551491-1 and lnc-ZNF674-1[44] Interestingly lncRNAs can be located in whole salivaHOTAIR is differentially expressed in saliva of metastaticoral squamous cell carcinoma patients compared to primarytumors These findings suggest that detection of lncRNAs in

saliva may be used as a noninvasive and rapid diagnostic toolfor diagnosis of oral carcinomas [4]

(ix) Bladder Oncogenic lncRNAs including UCA1 H19MALAT1 and linc-UBC1 (upregulated in bladder cancer1) are overexpressed in bladder carcinomas and activatePI3K-AKT and Wnt120573-catenin pathways A pilot study tookadvantage of this to evaluate potential application of UCA1in urinary sediments from patients with bladder carcinomasIt turned out to be especially valuable for superficial G2G3patients at a high risk for muscular invasion indicatingthat UCA1 may be a new promising urinary biomarker forthe diagnosis of bladder cancer H19 expression levels areremarkably increased in bladder carcinomas compared tonormal tissue and could also serve as another biomarkerMore recently a new lncRNA linc-UBC1 was found to beoverexpressed in 60 of invasive bladder carcinomas andcorrelated with lymph node metastasis and poor survivalMALAT1 is upregulated in bladder cancer and its expressionlevel is correlated with tumor grade and metastatic stage[34 35]

(x) Prostate Prostate cancer specific lncRNADD3PCA3 wasidentified fifteen years ago [86] PCA3 is a prostate-specificlncRNA markedly overexpressed in prostate carcinomas Itcan be detected in prostate cancer tissue urine andor urinesediments In recent studies PCA3 specificity was found evenhigher than prostate biomarker PSA and PCA3 score canaccurately predict tumor volume and pathological featureswhich may guide treatment [36 37] Therefore PCA3 canbe used as a noninvasive urine-based test for large-scalescreening protocols and for predicting prostate carcinomasaggressiveness This prominent example of rapid translationof lncRNAresearch into clinical practice offers a prototype fordeveloping different lncRNAs as biomarkers Other lncRNAssuch as PCGEM1 and PCAT1 (prostate cancer associatedncRNA transcript 1) are also prostate-specific posing asattractive biomarkers [38 39]

(xi)Melanoma BANCR (lncRNABRAF-activated noncodingRNA) plays a potentially functional role in melanoma cellsproliferation and migration by activating ERK12 and JNKMAPKpathwaysBANCR is upregulated in humanmalignantmelanoma and patients with high levels of expression have alower survival rate [41]

(xii) Hemopathies Several recent reports have revealed dereg-ulation of lncRNAs in leukemia including ANRIL lncRNA-P21 MEG3 Dleu2 HOTAIRM1 EGO and lncRNA-a7 More-overMEG3 UCA1 andH19 are upregulated in acutemyeloidleukemia [87 88]

(xiii) Ovary HOTAIR plays a pivotal role in epithelial ovariancancer (EOC) metastasis and could represent a novel prog-nostic marker and potential therapeutic target in patientswith EOC In recent study HOTAIR expression was elevatedin EOC tissues and its level of expression is highly positively


correlated with FIGO stage histological grade lymph nodemetastasis reduced overall survival and disease-free survival[33]

Because of their central role in genes expression regu-lation lncRNAs could also represent potential therapeutictargets Better characterization of lncRNAs (structure func-tions polymorphisms and intracellular topography) couldhelp in faster development of new anticancer strategiesmodulating expression levels and functions of deregulatedlncRNAs Targeting lncRNAs offers novel exciting opportu-nity to treat cancer Currently nucleic acid-based methodsprevail in targeting RNA by regulating levels of expres-sion and modifying their structures or mature sequencesAmong them RNA interference (RNAi) based techniquesare arguably the most popular methods to inhibit lncRNAsin cancer cells Both siRNAs and shRNAs exhibit greatRNA selectivity and knockdown efficiency Meanwhile otherestablished methods in inhibiting cancer-associated RNAincluding antisense oligonucleotide (ASO) ribozyme andaptamer are also effective to modulate lncRNAs and theyshow unique features that can have advantages over siRNAs[89]

In principle targeting of lncRNAs can be achieved usingthe several approaches including siRNA-mediated silencingfunctional block using small molecules or oligonucleotideinhibitors to prevent interactions of lncRNAs with proteinsand structure disruption via small molecules or oligonu-cleotide inhibitors to change or mimic their secondarystructure to compete for their binding partners [90]

(1) Small interfering RNAs (siRNA) are short stretched(19ndash30 nt) double-stranded RNAs that target RNAmoleculesvia complementary to unpaired lncRNA sequencesTheRNAduplex of siRNAs must be unwound into single strandsbefore assembling into the active RNA-induced silencingcomplex (RISC) SiRNAs are fully complementary to theirRNA target that is then cleaved at a single phosphodiesterbond located near the centre of the sequence complement tothe siRNA sequence [90] SiRNAs exhibit high knockdownefficiency to many oncogenic lncR-NAs in cancer cells andinduce anticancer effects both in vitro and in vivo Forinstance depletion of HOTAIR by siRNAs decreases matrixinvasiveness of breast cancer cells and inhibits tumor growthof pancreatic cancer xenograft HULC and MALAT1 siRNA-induced knockdown inhibits HCC cell proliferation andcell cycle progression [91] Furthermore siRNA-mediatedknockdown of H19 induces apoptosis and inactivates p53[92] Phase I and II clinical therapeutic trials have beenevaluated using siRNAs to inhibit critical cancer-associatedgenes including siRNA-EphA2-DOPC (targeting EphA2)TKM-080301 (targeting PLK1) and CALAA-01 (targetingRRM2)Therapy combining siRNA drug siG12D LODER andAtu027 with conventional chemotherapy is studied in phaseII for its therapeutic effect in advanced pancreatic cancerpatients However the main obstacle of siRNAs therapeuticsremaining is their delivery SiRNAs using ribonucleic acid asbuilding block are susceptible to be degraded by nuclease andhave poor pharmacokinetics

(2) Antisense oligonucleotides (ASO) are short single-stranded DNAs or RNAs (between 8 and 50 nt) designed

with sequence specific to target lncRNA [90] ASOs directlyhybridize to lncRNA transcripts via base pairing and endoge-nous RNase H1 which results in cleavage of lncRNAmolecules then recognizes hybrids formed ASOs modulatelncRNAs function through degradation of lncRNA tran-scripts Inhibition of MALAT1 by ASO attenuates variousmalignant phenotypes in cancer cells via cycle arrest incervical cancer cells [93] Injection of ASO into subcutaneoustumors of nude mice effectively inhibitsMALAT1 in vivo andblocks metastasis of lung cancer cells [91]

(3) Ribozymes are naturally produced RNA moleculesthat present intracellular catalytic functions One of theirfunctions is degradation of RNAmolecules Among all typesof ribozyme hammerhead ribozyme (HamRz) has caughtmajor interest as it shows good target inhibitory effect whilehaving the smallest RNA endoribonucleolytic motif as well asfunction independent to presence of metal ions [94] HamRzis single-stranded RNA in neutral condition and undergoesfolding in cells to expose the binding arms Binding ofHamRzto target sequence depends on complementary match withhomologous target site Both arms of HamRz have to bindwith target sites correctly in order to form functional catalyticmotif After binding HamRz catalyzes cleavage of the flankedRNA region downstream to a NUH site via destabilizingphosphodiester backbone of target RNA [90]

(4) Aptamers are short DNA or RNA oligonucleotidesor peptides that have a stable 3-dimensional structure invivo They have broad molecular targets including proteinRNA and small molecules that rely on fitting 3-dimensionalshape of their ligands [95] They specifically bind to theirtarget lncRNAs that rely on fitting 3-dimensional shape ofthe lncRNA structures Aptamers antagonize their lncRNAtargets by blocking the interactions between lncRNAs andcritical factors [90] Some reports show promising effects ofaptamers to either degrade RNA or inhibit RNA functionsimplying the potential of aptamers as therapeutic agents totarget lncRNAs Aptamers are used to modulate viral geneexpression by interacting with viral RNAs A hairpin aptameris identified to form stable and specific complex with thetransactivation response element (TAR) RNA element ofHIV-1 mRNA and decrease the TAR-dependent viral proteinexpression [96] Another aptamer selected against TAR ele-ment formed stable loopndashloop complexes with the elementto regulate the TAR-mediated process [97] Another studyshowed that aptamer could target the apical loop domainof pri-microRNA molecules and modulate the biogenesis ofmature microRNA [98]

(5) MicroRNAs (miRNAs) can interact with lncRNAsH19miR-675 signaling is critical in glioma progression Byanalyzing glioma gene expression data sets H19 is foundto be increased in high grade gliomas H19 depletion viasiRNA inhibits invasion in glioma cells Further H19 ispositively correlated with its derivate miR-675 expressionand reduction of H19 inhibits miR-675 expression MiR-675 modulates cadherin 13 expression by directly targetingits binding site within 31015840 UTR These results demonstratethat H19 regulates glioma development by deriving miR-675 and provide important clues for understanding keyroles of lncRNA-microRNA functional network in glioma


MicroRNA-based anticancer therapy has great potential asreports show there is apparent lack of adverse event in normaltissues when administrated with microRNA-based agentVarious microRNA delivery strategies such as cationic lipidand nanoparticle encapsulation are developed to improvemicroRNA shuttling into target cancer cells [40 99 100]The potential of microRNA regulation on lncRNAs canbe further realized by better understanding of intrageniclncRNA regulating elements

(6) Small molecules are synthesized to specifically bind toRNAbinding pockets of lncRNAsThey competewith proteinfactors or intracellular small ligands for binding lncRNAsBinding of small molecules may also induce conformationalchange within lncRNA molecules and disrupt formation ofimportant lncRNA structures [90]

3 Conclusion and Perspectives

Deregulated oncogenic and oncosuppressive lncRNAs areobserved at all stages of malignant tumors developmentof various origins Mechanisms implicating lncRNAs incarcinogenesis are dominated by deregulation of signalingpathways and altered epigenetic transcriptional and post-transcriptional expression of numerous genes Their use asbiomarkers and potential therapeutic targets could appearpromising

Major challenges of the next 10 years remaining are iden-tification mapping of all lncRNAs belonging to the humangenome and their functional characterization Althoughtheir mechanisms of action are better known it is stillpossible that lncRNAs are rather downstream products ofparticular chromatin structures or deregulated transcrip-tional processes Their functionality is currently still debatedbecause of their low level of expression and conservationbetween species Unlike proteins which often have well-defined functional areas it is currently impossible to predictlncRNAs function from their single sequence Howevermany traits exhibited by lncRNAs favor functionality includ-ing 31015840polyadenylation 51015840cap multiple exons transcriptionalactivation similar to that of mRNA K4K36 domain andalternative splicing These challenges will only be achievedthrough combined efforts of functional genomics epige-nomics and bioinformatics

Conflict of Interests

No potential conflict of interests was disclosed

Acknowledgment

This work was supported by Grant INCa-DGOS-4654

References

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[2] M K Iyer Y S Niknafs R Malik et al ldquoThe landscape oflong noncoding RNAs in the human transcriptomerdquo NatureGenetics vol 47 no 3 pp 199ndash208 2015

[3] R J Taft K C Pang T R Mercer M Dinger and J S MattickldquoNon-coding RNAs regulators of diseaserdquo Journal of Pathologyvol 220 no 2 pp 126ndash139 2010

[4] E A Gibb K S S Enfield G L Stewart et al ldquoLong non-coding RNAs are expressed in oral mucosa and altered in oralpremalignant lesionsrdquo Oral Oncology vol 47 no 11 pp 1055ndash1061 2011

[5] J M Perkel ldquoVisiting lsquoNoncodarniarsquordquo BioTechniques vol 54 no6 pp 301ndash304 2013

[6] K C Pang M C Frith and J S Mattick ldquoRapid evolution ofnoncoding RNAs lack of conservation does not mean lack offunctionrdquo Trends in Genetics vol 22 no 1 pp 1ndash5 2006

[7] O Wapinski and H Y Chang ldquoLong noncoding RNAs andhuman diseaserdquo Trends in Cell Biology vol 21 no 6 pp 354ndash361 2011

[8] S Ye L Yang X Zhao W Song W Wang and S ZhengldquoBioinformatics method to predict two regulation mechanismTFndashmiRNAndashmRNA and lncRNAndashmiRNAndashmRNA in pancre-atic cancerrdquo Cell Biochemistry and Biophysics vol 70 no 3 pp1849ndash1858 2014

[9] Q Jiang J Wang Y Wang R Ma X Wu and Y LildquoTF2LncRNA identifying common transcription factors for alist of lncRNA genes from ChIP-seq datardquo BioMed ResearchInternational vol 2014 Article ID 317642 5 pages 2014

[10] C Park N Yu I Choi W Kim and S Lee ldquolncRNAtor acomprehensive resource for functional investigation of longnon-coding RNAsrdquo Bioinformatics vol 30 no 17 pp 2480ndash2485 2014

[11] Z X Chen D Sturgill J Qu et al ldquoComparative validation ofthe D melanogaster modENCODE transcriptome annotationrdquoGenome Research vol 24 no 7 pp 1209ndash1223 2014

[12] Z Chen J Li L Tian et al ldquoMiRNA expression profile reveals aprognostic signature for esophageal squamous cell carcinomardquoCancer Letters vol 350 no 1-2 pp 34ndash42 2014

[13] M Hajjari M Behmanesh M Sadeghizadeh and M Zein-oddini ldquoUp-regulation of HOTAIR long non-coding RNA inhuman gastric adenocarcinoma tissuesrdquoMedical Oncology vol30 no 3 article 670 2013

[14] F Yang X Xue J Bi et al ldquoLongnoncodingRNACCAT1whichcould be activated by c-Myc promotes the progression of gastriccarcinomardquo Journal of Cancer Research and Clinical Oncologyvol 139 no 3 pp 437ndash445 2013

[15] W Sun Y Wu X Yu et al ldquoDecreased expression of longnoncoding RNA AC0966551-002 in gastric cancer and itsclinical significancerdquo Tumor Biology vol 34 no 5 pp 2697ndash2701 2013

[16] B Xiao and J Guo ldquoLong noncoding RNAAC0966551-002 hasbeen officially named as gastric cancer-associated transcript 1GACAT1rdquo Tumor Biology vol 34 no 5 p 3271 2013

[17] D Mei H Song K Wang et al ldquoUp-regulation of SUMO1pseudogene 3 (SUMO1P3) in gastric cancer and its clinicalassociationrdquoMedical Oncology vol 30 no 4 article 709 2013

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[81] R Backofen and T Vogel ldquoBiological and bioinformaticalapproaches to study crosstalk of long-non-coding RNAs andchromatin-modifying proteinsrdquo Cell and Tissue Research vol356 no 3 pp 507ndash526 2014

[82] M-C Lai Z Yang L Zhou et al ldquoLong non-coding RNAMALAT-1 overexpression predicts tumor recurrence of hepato-cellular carcinoma after liver transplantationrdquo Medical Oncol-ogy vol 29 no 3 pp 1810ndash1816 2012

[83] Z Yang L Zhou L-MWu et al ldquoOverexpression of long non-coding RNA HOTAIR predicts tumor recurrence in hepatocel-lular carcinomapatients following liver transplantationrdquoAnnalsof Surgical Oncology vol 18 no 5 pp 1243ndash1250 2011

[84] L Quagliata M S Matter S Piscuoglio et al ldquoLong noncodingRNA HOTTIPHOXA13 expression is associated with diseaseprogression and predicts outcome in hepatocellular carcinomapatientsrdquo Hepatology vol 59 no 3 pp 911ndash923 2014


[85] S-X Yuan F Yang Y Yang et al ldquoLong noncoding RNA asso-ciated withmicrovascular invasion in hepatocellular carcinomapromotes angiogenesis and serves as a predictor for hepato-cellular carcinoma patientsrsquo poor recurrence-free survival afterhepatectomyrdquo Hepatology vol 56 no 6 pp 2231ndash2241 2012

[86] M J G Bussemakers A Van Bokhoven G W Verhaegh etal ldquoDD3 a new prostate-specific gene highly overexpressed inprostate cancerrdquoCancer Research vol 59 no 23 pp 5975ndash59791999

[87] M Hajjari A Khoshnevisan and Y K Shin ldquoLong non-coding RNAs in hematologic malignancies road to transla-tional researchrdquo Frontiers in Genetics vol 4 article 250 2013

[88] E F Heuston K T Lemon and R J Arceci ldquoThe beginningof the road for non-coding RNAs in normal hematopoiesis andhematologic malignanciesrdquo Frontiers in Genetics vol 2 article94 2011

[89] C H Li and Y Chen ldquoTargeting long non-coding RNAsin cancers progress and prospectsrdquo International Journal ofBiochemistry and Cell Biology vol 45 no 8 pp 1895ndash1910 2013

[90] L Ma M-S Chua O Andrisani and S So ldquoEpigeneticsin hepatocellular carcinoma an update and future therapyperspectivesrdquo World Journal of Gastroenterology vol 20 no 2pp 333ndash345 2014

[91] T Gutschner M Hammerle and S Diederichs ldquoMALAT1mdashaparadigm for long noncoding RNA function in cancerrdquo Journalof Molecular Medicine vol 91 no 7 pp 791ndash801 2013

[92] F Yang J Bi X Xue et al ldquoUp-regulated long non-coding RNAH19 contributes to proliferation of gastric cancer cellsrdquo FEBSJournal vol 279 no 17 pp 3159ndash3165 2012

[93] V Tripathi Z Shen A Chakraborty et al ldquoLong noncodingRNA MALAT1 controls cell cycle progression by regulatingthe expression of oncogenic transcription factor B-MYBrdquo PLoSGenetics vol 9 no 3 Article ID e1003368 2013

[94] D E Ruffner G D Stormo and O C Uhlenbeck ldquoSequencerequirements of the hammerhead RNA self-cleavage reactionrdquoBiochemistry vol 29 no 47 pp 10695ndash10702 1990

[95] J Muller O El-Maarri J Oldenburg B Potzsch and GMayer ldquoMonitoring the progression of the in vitro selection ofnucleic acid aptamers by denaturing high-performance liquidchromatographyrdquo Analytical and Bioanalytical Chemistry vol390 no 4 pp 1033ndash1037 2008

[96] G Kolb S Reigadas D Castanotto et al ldquoEndogenous expres-sion of an anti-TAR aptamer reduces HIV-1 replicationrdquo RNABiology vol 3 no 4 pp 150ndash156 2006

[97] M Watrin F von Pelchrzim E Dausse R Schroeder and J-J Toulme ldquoIn vitro selection of RNA aptamers derived from agenomic human library against the TAR RNA element of HIV-1rdquo Biochemistry vol 48 no 26 pp 6278ndash6284 2009

[98] C E Lunse G Michlewski C S Hopp et al ldquoAn aptamertargeting the apical-loop domain modulates pri-miRNA pro-cessingrdquo Angewandte ChemiemdashInternational Edition vol 49no 27 pp 4674ndash4677 2010

[99] Y Wu Y Xiao X Ding et al ldquoA mir-200b200c429-bindingsite polymorphism in the 31015840 untranslated region of the AP-2120572gene is associated with cisplatin resistancerdquo PLoS ONE vol 6no 12 Article ID e29043 2011

[100] J Wu G Wu L Lv et al ldquoMicroRNA-34a inhibits migrationand invasion of colon cancer cells via targeting to Fra-1rdquoCarcinogenesis vol 33 no 3 pp 519ndash528 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Type and main function of no-coding RNAs

Type Size (nt) FunctionsShort ncRNAs 19ndash31

miRNAs Targeting of mRNAs regulation of proliferation differentiation and apoptosisinvolved in human development

siRNAs Posttranscriptional gene silencing defense against pathogenic nucleic acidstiRNAs Regulation of transcription by targeting epigenetic silencing complexes

piRNAs Transposon repression DNA methylation development of germ cell stemself-renewal and retrotransposon silencing

tel-sRNAs Epigenetic regulationMid-size ncRNAs le200

snoRNAs rRNA modificationsPASRs Regulation of the transcription of protein-coding genesTSSa-RNAs Maintenance of transcriptionPROMPTs Activation of transcriptioncrasiRNAs Recruitment of heterochromatin andor centromeric proteins

Long ncRNAs gt200lincRNAs Involvement in biological processes such as dosage compensation andor imprintingIntronic lncRNAs Possible link with posttranscriptional gene silencing

T-UCRs Regulation of miRNA and mRNA levels and antisense inhibitors for protein-codinggenes or other ncRNAs

TERRAs Negative regulation of telomere length and activity through inhibition of telomerasePseudogene RNAs Regulation of tumor suppressors and oncogenes by acting as microRNA decoyslncRNAs with dual functions Modulate gene expression through diverse mechanisms

summarize recent literature data on lncRNAs their involve-ment in carcinogenesis and their value as biomarkers andpotential therapeutic targets

13 Definition of lncRNAs LncRNAs transcripts are definedby a length greater than 200 nts without any potential oftranslation [6] This definition is however arbitrary andcannot now be based on a set of physical biochemicalstructural or functional criteria Length is not an absolutecriterion because ncRNAs smaller than 200 nts (BC1 snaR)are included in the lncRNAs subgroup There is no specifictranscription of lncRNAs the transcriptional machinerybeing common to lncRNAs and mRNAs Lack of readingframe cannot also be a discriminating factor betweenmRNAsand ncRNAs because 50 of lincRNAs contain regions withhigh translational potential comparable to those of mRNAssuggesting that they are actively exported to cytoplasm andtranslated into short polypeptides albeit certainly not activeNoncoding characteristic is then not specific to lncRNAs asbifunctional coding and noncoding lncRNAs and lncRNAscontaining other ncRNAs (miRNAs snoRNAs) have recentlybeen discovered Several mRNAs lose their ability to encodeproteins (Xist) while others acquire coding function (SRA)[7] Furthermore by incorporating multiple exons fromcoding and noncoding genes alternative splicing generatesambiguous transcripts beyond all classifications Neverthe-less this definition appears currently to be the best that couldbe used waiting for a functional characterization of lncRNAs

14 Investigative Methods and Annotation of lncRNAs Thefirst lncRNAs were discovered by using functional geneticapproaches based on their relation to specific cellular mecha-nisms (i) Ubxn transcriptional interference-induced repres-sion by trithorax-promoted lncRNAbdx (ii) sex chromosomedosage compensation and Xist-induced X chromosomeand (iii) inactivation and genomic imprinting promoted byantisense lncRNAs Airn and Kcnq1 These seminal studiesestablished the concept of functional lncRNAs acting astranscriptional regulators involved in important and diversebiological processes Over the past two decades systematicglobal sequencing of cDNA libraries yielded comprehensiveknowledge of the transcribed but noncoding component ofthe genome These transcriptomic analyses have identifiednumerous lncRNAs RNA immunoprecipitation and siRNA-induced inactivation techniques led to functional validationof many lncRNAs

Currently lncRNAs identification and functional charac-terization are based on an experimental approach combining(i) discovery of new noncoding transcripts using RNA-seqand ChIP-seq (ii) annotation of transcribed regions extentand quantity of products transcribed through bioinformaticanalysis (iii) lncRNAs quantification in various cellular andtissue types and conditions (iv) coexpression or coregulationstudies and (v) gain and loss functional tests on cell linesand xenografts (siRNAs ASO TALEN and CRISPRCAS9)Advantage of RNA-seq technique resides in its sensibilityallowing most weakly expressed lncRNAs to be identified













































Geneexpression

lncRNAs


lncRNA

Protein


lncRNA


lncRNAs


































































Acknowledgment


References















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























































Geneexpression

lncRNAs


lncRNA

Protein


lncRNA


lncRNAs


































































Acknowledgment


References















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































































Acknowledgment


References















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article long noncoding rnas as new...

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