LncRNA: A link between RNA and cancer

    LncRNA: A link between RNA and cancer Guodong Yang, Xiaozhao Lu, Lijun Yuan PII: DOI: Reference:

S1874-9399(14)00230-2 doi: 10.1016/j.bbagrm.2014.08.012 BBAGRM 807

To appear in:

BBA - Gene Regulatory Mechanisms

Received date: Revised date: Accepted date:

9 May 2014 4 August 2014 18 August 2014

Please cite this article as: Guodong Yang, Xiaozhao Lu, Lijun Yuan, LncRNA: A link between RNA and cancer, BBA - Gene Regulatory Mechanisms (2014), doi: 10.1016/j.bbagrm.2014.08.012

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ACCEPTED MANUSCRIPT LncRNA: a link between RNA and cancer

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Guodong Yang1*,§, Xiaozhao Lu2*, Lijun Yuan3,§

The State Key Laboratory of Cancer Biology, Department of Biochemistry and

Molecular Biology, the Fourth Military Medical University, Xi’an 710032, PR China. Department of Nephrology, 323 Hospital of PLA, Xi'an 710054, PR China

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Tangdu Hospital, the Fourth Military Medical University, Xi’an 710038, PR China

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*

To whom correspondence should be addressed:

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These authors contributed equally to this work.

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Guodong Yang, NO.17 Changlexi Road, the Fourth Military Medical University, 710032, Xi’an PR China, email: [email protected], tel: +86-29-84774513, fax

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+86-29-84773947.

Lijun Yuan, NO.1 Xinshi Road, Tangdu Hospital, the Fourth Military Medical University, 710038, Xi’an PR China, email: [email protected], tel: +86-29-84777171, fax +86-29-84777471.

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ACCEPTED MANUSCRIPT Abstract Unraveling the gene expression networks governing cancer initiation and development is essential

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while remains largely uncompleted. With the innovations in RNA-seq technologies and computational

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biology, long noncoding RNAs (lncRNAs) are being identified and characterized at a rapid pace. Recent findings reveal that lncRNAs are implicated in serial steps of cancer development. These lncRNAs interact with DNA, RNA, protein molecules and/or their combinations, acting as an essential

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regulator in chromatin organization, transcriptional and post-transcriptional regulation. Their

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misexpression confers the cancer cell capacities for tumor initiation, growth, and metastasis. The review here will emphasize their aberrant expression and function in cancer, and the roles in cancer

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diagnosis and therapy will be also discussed.

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Keywords：long non-coding RNAs; cancer; epigenetics; transcriptional regulation; posttranscriptional

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regulation; cancer diagnosis and therapy

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ACCEPTED MANUSCRIPT 1. Introduction Ever since the proposal of central dogma of molecular biology in 1961[1], RNA was considered as an

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intermediate between DNA and protein. The central dogma has provided us a simplified framework of

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how genetic information is translated into diversity of biological process. Later on, these intermediate RNAs (mRNAs) are found to be just a small fraction of the total RNA population, as the discovery of non-coding RNAs (ncRNAs). These ncRNAs function directly as structural, catalytic or regulatory

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RNAs, rather than encoding proteins [2-4]. Up to now, there are still no satisfactory classifications for

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these transcripts. Based on the expression and function, ncRNA can be classified as groups including ‘housekeeping’ ncRNAs (ribosomal RNA, transfer RNA, small nuclear RNA and small nucleolar

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RNA), some lowly expressed regulatory ncRNAs and several other poorly characterized types of

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ncRNAs[5]. According to their sizes, the regulatory ncRNAs can be further classified as small ncRNAs

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(< 200 bps, e.g. miRNAs, siRNAs, and piRNAs) and long ncRNAs (lncRNAs) (>200 bps, e.g. lincRNAs, macroRNAs)[5].

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During the past decades of RNA biology study, multiple lncRNAs have been identified, such as Xist [6] and H19 [7], which hold as milestones in lncRNA biology. With the advent of advanced sequencing technologies and findings from large-scale consortia focused on characterizing functional genomic elements, such as ENCODE (encyclopedia of DNA elements), more and more lncRNAs are being identified and awaited for functional validation. According to the recent data by ENCODE Project Consortium in 2012, there are about 9,640 long non-coding RNA (lncRNA) loci in human genome [8, 9], while the number continues to grow. All of these have shed light on the promising future of lncRNA study. LncRNAs have been found to be involved in the regulation at chromatin organization, transcriptional, and post-transcriptional levels[10], revolutionizing our understanding of the 3

ACCEPTED MANUSCRIPT architecture, activity and regulation of the eukaryotic genome. LncRNAs have added another layer of genome complexity; meanwhile they provide alternative explanation that the diversity of biology is not

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solely on the protein coding genes, their splicing or posttranslational regulation.

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LncRNAs have emerged as an essential regulator in almost all aspects of biology. Accumulating evidence suggests that lncRNAs play an important role in tumorigenesis [11]. In this review, we will briefly review the structure and function of lncRNAs, and then emphasize their aberrant expression and

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their functional roles in cancer development, diagnosis and therapy.

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ACCEPTED MANUSCRIPT 2. LncRNA: an emerging star in ncRNA world 2.1 Genomic distribution of lncRNA and their expression.

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Before we discuss the role of lncRNAs in cancer, we first need to refer their structure, expression and

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function under physiological conditions. According to a recent manual annotation of lncRNAs, there should be about 9640 lncRNAs, approximately half of the protein encoding genes[8, 9] According to LNCipedia 2.0, the latest version of this long non-coding RNA database, there are already 32,183

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human annotated lncRNAs. Currently, few lncRNAs are functionally validated[12]. LncRNAs are

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transcribed from intergenic regions, intragenic regions, or from specific chromosomal regions. These intergenic lncRNAS are called large intergenic ncRNAs (lincRNAs), and these lincRNAs should be 5

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kb away from protein-coding genes[13]. For the lncRNA transcribed from intragenic regions, they can

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be further classified as in antisense, overlapping, intronic, bidirectional orientations relative to

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protein-coding genes or gene regulatory regions, such as UTRs, promoters and enhancers (see review[14]). Almost 40% (3934 lncRNA genes, 5361 transcripts) of GENCODE lncRNAs intersect

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protein-coding gene loci [9].

LncRNA expression is controlled by both transcriptional and epigenetic factors. Active histone marks correlate well with the expression of lncRNA genes, which is similar to the protein coding genes. However, lncRNA genes are found to harbor higher methylation density around TSS (transcriptional start site) than protein-coding genes regardless of their expression status, suggesting that epigenetic regulation of lncRNAs at the level of DNA methylation is markedly dissimilar[15]. Up to now, multiple transcriptional factors are found to transactivate the expression of lncRNAs, including Nanog, NFkappaB, Sox2, Oct4 (also known as Pou5f1), tumour protein 53 (TP53)[13, 16] and zinc finger protein 143 (ZNF143)[17]. Transcribed lncRNAs are further subject to post-transcriptional 5

ACCEPTED MANUSCRIPT processing[8, 9], such as 5’capping, polyadenylation, alternative splicing, RNA editing[18]. Specifically, lncRNAs show a relatively lower expression level but much more tissue-specific pattern

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than protein-coding genes [9], suggesting a distinguished and regulatory role of lncRNAs.

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It is important to note that there is no strict dichotomy between protein-coding and non-coding transcripts. Some ncRNAs contain open reading frames and can be translated[19]. On the other hand, some protein encoding RNAs, such as the tumor protein 53 (TP53) and steroid receptor RNA activator

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mRNA, could also involve in meaningful cellular regulatory and functional processes, in addition to

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the routine role for translation[20, 21].

2.2 lncRNA: an expanding modulator in gene regulation

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Different from the intensively studied miRNA, lncRNA is large and thus has a complex secondary and

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tertiary structure. The complicated structure endows lncRNAs with the abilities to bind protein, RNA

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and/or DNA partners and thus with several regulatory capacities (Fig 1). They act as activators, decoys, guides, or scaffolds for their interacting proteins, such as transcription factors and histone modifiers

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(Fig 1a-d). Besides these transcriptional and epigenetic regulation, lncRNAs have also been found to be important players in posttranscriptional regulation, such as mRNA editor, mRNA splicing regulators, and reservoirs of small ncRNAs (Fig 1e-k). Breakthroughs over the past few years have revealed numerous examples of lncRNAs in orchestrating the regulation from DNA to RNA to protein (examples can be seen in Table 1). Some of the recent excellent reviews have summarized the mechanisms how lncRNA regulates the chromatin structure and transcriptional control [5, 22, 23]. We here will mainly focus on the mechanism how lncRNAs regulating mRNA biogenesis. 2.2.1 LncRNA: regulator of mRNA processing Alternative splicing and editing contribute to increasing gene isoform diversity. In some cases, 6

ACCEPTED MANUSCRIPT lncRNAs have an antisense orientation to known protein-coding genes and thus can bind these pre-mRNAs. The double-stranded RNA could recruit ADAR (adenosine deaminase acting on RNA)

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enzymes. ADAR in turn catalyze adenosine to inosine conversion in double-stranded RNA, and this

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conversion can influence RNA structure, splicing patterns, coding potential (Fig 1e). Given that many lncRNAs at least partially overlap with their sense mRNA, the potential for double-stranded RNA editing substrates is extensive. However, currently there are few convincing samples of this regulation.

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However, both mRNA sand lncRNAs have been found to be edited by ADAR[24], it is thus reasonable

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to anticipate that lncRNAs are likely to help diversify the transcriptome and proteome through control of RNA editing. Besides mRNA editing, lncRNAs can also regulate mRNA alternative splicing. For

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example, a lncRNA which has the antisense orientation to ZEB2 can bind the ZEB2 pre-mRNA,

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preventing the splicing of a 5’ UTR IRES-containing intron, thus increases protein levels of ZEB2(Fig

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1f)[25]. Besides this lncRNA:RNA interaction mediated alternative splicing, lncRNAs can also interact with splicing factors and thus change the splicing profile. Interaction between MALAT1 and the

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serine/arginine (SR) splicing factors influences the distribution and phosphorylation of SR, resulting in changes of the alternative splicing of a set of endogenous pre-mRNAs (Fig 1f) [26]. 2.2.2 LncRNAs: reservoir of small ncRNAs Integrative transcriptome analysis suggest that a subset of long non-coding RNAs could be processed to small RNAs (Fig 1g) [27]. LncRNA H19 itself harbors the miR-675, and both H19 and the miR-675 are involved in the

regulation of osteoarthritis[28]. Similarly, lncRNA LOC554202 could encode

miR-31, and promoter methylation of lncRNA LOC554202 leads to decreased miR-31 and thus contributes to breast cancer invasion and metastasis[29]. Besides miRNAs, lncRNA was also found to produce other small ncRNAs. MALAT1 may further serve as a precursor to a 61-bp, tRNA like 7

ACCEPTED MANUSCRIPT ncRNA[30], though the detailed biological function remains unclear. All of these experimental studies confirm that some lncRNAs serve as a reservoir of small ncRNAs and could have a dual regulatory

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output.

2.2.3 lncRNA: scaffold for lncRNA-mRNA/miRNA interaction

Aside from harboring the small ncRNAs,, some studies describe interactions between miRNAs and

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lncRNAs, with lncRNAs acting either as miRNA sponges (Fig 1h)[31] or just direct targets of

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miRNAs[32]. For example, highly up-regulated in liver cancer (HULC) may act as an endogenous ‘sponge’, which down-regulates a series of miRNAs activities, including miR-372[33]. PTENP1

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(PTEN pseudogene 1) harbors similar 3’UTR with PTEN and thus sequesters those miRNAs which are

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supposed to target PTEN [34, 35]. Through this, PTENP acts as a decoy to attenuate miRNA mediated

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downregulation of PTEN. While miR-671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration-Related protein 1 (CDR1) locus in an Ago2-slicer-dependent manner, which

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in turn results in a decrease of this lncRNA[36], providing the evidence for non-coding antisense transcripts as functional miRNA targets. Similarly, association of the RNA-binding protein HuR with lincRNA-p21 favored the recruitment of let-7/Ago2 to lincRNA-p21, leading to lower lincRNA-p21 stability[37]. Based on the abundance of miRNAs and lncRNAs in cells, miRNA:lncRNAs interaction should be universal and functionally important. Recently, an online software named miRcode is designed to search a possible interaction between a lncRNA and microRNA of interest[32], and thus would promote the study in this field. β-secretase-1

(BACE1),

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critical

enzyme

in

Alzheimer's disease

pathophysiology.

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BACE1-antisense transcript is markedly up-regulated in brain samples from Alzheimer's disease 8

ACCEPTED MANUSCRIPT patients. BACE1-antisense transcript competes for binding within the same region in the open reading frame of the BACE1 mRNA with miR-485-5p. By masking the binding site for miR-485-5p,

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prevents miRNA-induced repression of BACE1 mRNA [38](Fig 1i).

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BACE1-antisense transcript acts as a miRNA blocker and thus the increase of BACE1-antisense

Besides competing for miRNA/mRNA binding with their coding counterparts/miRNAs, lncRNAs could also target mRNAs directly. As we know, Staufen 1 (STAU1, a protein that binds to

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double-stranded RNA)-mediated messenger RNA decay (SMD) involves the degradation of

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translationally active mRNAs by binding the target mRNAs in the 3'-untranslated regions (3' UTRs). Recently, a kind of lncRNAs named half-STAU1-binding site RNAs (1/2-sbsRNAs), can imperfectly

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base-paired with the Alu element in the 3' UTR of SMD targets and thus form STAU1-binding sites,

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inducing target mRNA decay (Fig 1j) [39]. With many of those antisense lncRNA transcripts

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anticipated to form duplex with their sense counterparts, lncRNAs are likely to promote mRNA degradation ubiquitously. LncRNA:mRNA interaction not only alter the target mRNA stability, but also

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affect the translational efficiency. LincRNA-p21 could interact with JUNB and CTNNB1 mRNAs and selectively lower their translation [37]. In contrast, some other lncRNAs could also increase the target mRNA translational efficiency. Ubiquitin carboxy-terminal hydrolase L1 (Uchl1) is a gene involved in brain function and neurodegenerative diseases. Antisense Uchl1 is a 5′ head-to-head transcript that initiates within the second intron of Uchl1 and overlaps the first 73 nucleotides of the sense mRNA including the AUG codon, while the non-overlapping part of the transcript contains two embedded repetitive sequences, SINEB1 of the F1 subclass (Alu) and SINEB2 of the B3 subclass. Upon stress, antisense Uchl1 increases UCHL1 protein synthesis at a post-transcriptional level. Antisense Uchl1 activity depends on the presence of a 5' overlapping sequence and an embedded inverted SINEB2 9

ACCEPTED MANUSCRIPT element [40, 41](Fig 1k). Regarding that the SINE and overlapping sequence features are shared by other natural antisense transcripts, it is anticipated that this kind of regulation might be intensively

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involved in translational control.

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In view of the above examples, we can see that lncRNAs contribute to nearly all the aspects of mRNA processing and posttranscriptional regulation. And the interaction among lncRNAs/miRNAs/mRNAs basically depends on the base:base pairing rule, suggesting that lncRNAs function as a big player in

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gene regulation.

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Notably, RNA binding proteins are one of the most important determinants in posttranscriptional regulation, which play a fundamental role in nearly all aspects from mRNA editing to degradation. The

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interaction among lncRNAs/miRNAs/mRNAs and the subsequent biological effects can’t be completed

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without RNA binding proteins, such as the above mentioned ADAR, splicing factors, Staufen, HuR and

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so on. LncRNAs are found to interact with other RNA binding proteins, changing the activity of RNA binding proteins.

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For example, after induction by DNA damage, long ncRNAs associated with the cyclin D1 gene promoter could bind to and modulate the activities of the RNA binding protein TLS (translocated in liposarcoma) by an allosteric function. TLS subsequently inhibits the histone acetyltransferase activities of CREB binding protein and p300 to silence cyclin D1 expression[42]. Study on the lncRNAs and their complicated interaction networks are just beginning, and future identification of lncRNA-protein interaction would of course expand this list. Taken together, we can see that LncRNAs serve as a platform for DNA:RNA, RNA:RNA, RNA:protein interactions or their combinations, which endows the lncRNAs an essential role in multiple biological processes.

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Table 1 Examples of how lncRNA serves as an essential component and platform for complicated interactions. Examples

Functions

lncRNA :Chromatin regulators

SRA:CTCF

Enhances insulator function of CTCF

[43]

HOTAIR :LSD1-CoREST

Targets the LSD1 complex to demethylate H3K4me2

[44]

Xist :PRC2

Targets PRC2 either in cis or trans to mediate H3K27 methylation

[45]

Gas5:Glucocorticoid receptor

Titrates GR away from target genes

[46]

PANDA: NF-YA

Titrates NF-YA away from apoptotic genes

[47]

H19( miR-675)

Produces miR-675

[28]

LOC554202(miR-31)

Produces miR-31

[29]

HULC:miR-372

Competes for binding miR-372 with protein coding counterparts

[33]

CDR1-AS:miR-671 PTENP:miR20/19 NAT-ZEB2

[36]

Competes with PTEN for binding with miRNAs

[35]

Alternative splicing Induces degradation of target mRNA

[37]

1/2-sbsRNAs:SMD mRNA

Induces degradation of target mRNA

[39]

LincRNA-p21:JUNB

Induces degradation of target mRNA

[37]

LincRNA-p21:HuR

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LincRNA-p21:JUNB

lncRNA:RNA binding protein

References

Degrades CDR1-AS

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lncRNA:mRNA

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lncRNA(miRNA)

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lncRNA:TFs

CR

Category

Induces degradation of lincRNA-p21

[37]

ncRNACCND1:TLS

Allosterically binds TLS and changes its activity

[33]

MALAT1：serine/arginine (SR)

Modulates SR splicing factor phosphorylation and thus the downstream

[26]

proteins

target splicing

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ACCEPTED MANUSCRIPT 3. LncRNAs in cancer In a molecular perspective, cancer is a genetic disease due to aberrant expression and function of tumor

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suppressor and oncogenic genes. Besides the canonical protein encoding genes, more and more

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lncRNAs are found to be oncogenes or tumor suppressors, adding a new layer of complexity to the molecular architecture of human cancers (Table 2). Here we will focus on how these lncRNAs are aberrantly expressed in cancers and their contribution to cancer hallmarks.

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3.1 Deregulation of lncRNAs in cancer

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As discussed earlier, lncRNAs are processed similarly as the mRNA. Malignant transformation is the consequence of a multistep process involving different genetic and epigenetic changes in numerous

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genes in combination with host genetic background and environmental factors. Up to now, genetic,

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epigenetic and transcriptional regulatory mechanisms have been clarified to involve in lncRNA

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deregulation in cancers and dozens of lncRNAs have been identified deregulated. 3.1.1 Deregulation of lncRNA due to genetic and epigenetic changes

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Over the past decade multiple studies have identified small- and large-scale mutations affecting noncoding regions of the genome, including chromosomal translocations, copy-number alterations, nucleotide expansions, and single nucleotide polymorphisms (SNPs)[48]. Previously, the variation occured outside of protein-coding genes are often disregarded. The emerging studies are starting to link distinct types of mutations in lncRNA genes with cancer, and with such an effort, we will build a robust landscape of lncRNA in cancer and add a new layer for comprehensive understanding of cancer. Copy number alteration (CNA), which is associated with genetic instability, represents molecular disorders acquired by cancer cells during neoplastic transformation. Recently, a focal region of chr3q13.31 (osteo3q13.31) harboring CNAs in 80% of osteosarcomas and most (67%) osteo3q13.31 12

ACCEPTED MANUSCRIPT CNAs are deletions, with 75% of these monoallelic and frequently accompanied by loss of heterozygosity (LOH) in flanking DNA. Notably, these CNAs often involve the noncoding RNAs

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LOC285194 and BC040587. Further study revealed that LOC285194 acts as a tumor suppressor and

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genetic deletions of LOC285194 is also associated with poor survival of osteosarcoma patients[49]. In addition to LOC285194, loss of heterozygosity (LOH) of the maternal allele of KCNQ1OT1 is also seen in cancers[50]. Gene deletion of PTENP was also seen in melanoma[51] and gain of chromosome

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17q leads to overexpression of oncogenic ncRAN in neuroblastoma[52]. However, while established

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protein-coding oncogenes and tumor suppressors often display striking patterns of focal DNA copy-number alteration in tumors, similar evidence is largely lacking for lncRNAs. Especially their

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roles in cancer development are hard to tell, as complicated by the role of their adjacent sense protein

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coding genes. Recently, Akrami R et al examined the regions of focal copy-number change that lack

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protein-coding targets and identified an intergenic lncRNA on chromosome 1, OVAL, that shows narrow focal genomic amplification in a subset of tumors, providing evidence that lncRNAs themselves

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might independently contribute to cancer development[53]. In fact, many lncRNAs are located in the loci sensitive to number variations in cancer. And the elucidation of the involvement of lncRNAs in cancer is only in its infancy and large numbers of lncRNAs related genetic changes await clarification [54, 55]. We can expect an expanding list of the lncRNAs following this category. Besides the genetic instability, SNP is also found in the lncRNAs and might contribute to the aberrant expression of lncRNAs in cancer. In thyroid cancer patients, the polymorphism rs944289 in the PTCSC3 promoter region destroys its response to C/EBPα and C/EBPβ and thus results in the deregulation[56]. In prostate cancer, the SNPs between rs1456315 and rs7463708 are associated with cancer susceptibility and expression of the oncogenic lncRNA PRNCR1[57]. In liver cancer, variant 13

ACCEPTED MANUSCRIPT genotypes of rs7763881 in HULC may contribute to decreased susceptibility to HCC in HBV persistent carriers[58]. Very recently, oncogenic HOTAIR rs920778 TT carriers have found to have a 1.37-fold,

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1.78-fold and 2.08-fold increased esophageal squamous cell carcinoma (ESCC) risk in varied

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populations when compared with the these rs920778 CC carriers, which might be explained by enhancer in this region[59].These above SNP mainly affects the regulatory regions of lncRNAs and thus change the absolute expression. Previous finding on miRNAs showed that the SNP or mutation of

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miRNAs leads to functional changes and thus is involved in carcinogensis[60], raising the possibility

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that SNP and point mutations in lncRNAs . It has been challenging to determine the contribution of small mutations in lncRNAs themselves to cancer, because we have yet to characterize how the

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alteration of the lncRNA sequence affects their interaction with others, such as DNA, protein and other

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RNAs[48].

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Epigenetic changes occur both in the initiation and progression of cancers. Multiple lncRNAs have been found to be misexpressed in cancers due to epigenetic changes. As we know, the epigenetic

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regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development. There are a whole bunch of lncRNAs localized in the imprinted loci, playing an important role in the imprinted loci formation, maintenance[61]. To this end, these lncRNAs are epigenetically susceptible to epigenetic activation or inactivation. One of such examples is H19 de-repressed by Mineral dust-induced gene (Mdig). Mdig down-regulates the H3K9me3 and heterochromatin in the H19 loci and thus increase the oncogenic H19[62]. Other examples of the defect of imprinting related increase of lncRNAs include but not limit to KCNQ1OT1 in multiple cancers [50, 63-65]. In contrast, high CpG methylation of the promoter could lead to the tumor suppressive lncRNA MEG3 [66] and LOC554202[29] downregulation. 14

ACCEPTED MANUSCRIPT 3.1.2 Deregulation of lncRNAs due to other regulatory mechanisms Besides the genetic and epigenetic changes conferring the misexpression of lncRNAs in cancer, there

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are dozens of lncRNAs altered in cancers have been documented to be regulated by specific oncogenic

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and tumor-suppressor related signals and regulatory factors. TGF-β signal could transcriptionally activate lncRNA-ATB (lncRNA-activated by TGF-β) in hepatocellular carcinoma (HCC) and the latter in turn contributes to the metastasis[67]. Similar as the HIF1a, aHIF, HIF1A antisense RNA 2, has been

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shown to be regulated by hypoxia, which in turn induces the angiogenesis in types of cancers, such as

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breast and renal cancer[68-72]. In addition to aHIF, the imprinted H19 is also found to be upregulated by the oncogenic stimuli hypoxia[73]. H19 could be also transcriptionally activated by Myc, which

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leads to increased expression in breast and lung carcinomas[74]. In liver cancer, the highly expressed

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HULC could be due to the oncogenic HBx[75] and CREB[33], and the increased lncRNA-HEIH could

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be induced by Sp1[37]. In contrast, the tumor suppressor p53 could also activate a bundle of lncRNAs[13], and tumor suppressive linc-p21 is one of these mostly studied in cancers[16]. Recently,

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MEG3 has been identified to be regulated by miR-29, although in an indirect way[76]. A genomic wide analysis revealed that there are many specific lncRNAs are transcriptionally regulated by key transcription factors such as p53, NFkappaB, Sox2, Oct4 (also known as Pou5f1) and Nanog[13]. Most of these transcriptional factors are correlated with cancers, and their contribution to the misexpressed lncRNAs is worth of further examination. As discussed before, lncRNA expression regulation shares great similarity with the protein coding genes, the regulatory mechanism found in protein coding genes would be possibly involved in the regulation of lncRNAs. However, attention should be also paid to the unique parts of lncRNAs, such as the degradation of these non-coding lncRNAs during cancer development. Association of the RNA-binding protein HuR with lincRNA-p21 favored the recruitment 15

ACCEPTED MANUSCRIPT of let-7/Ago2 to lincRNA-p21, lowering lincRNA-p21 stability[37], further studies are needed to test whether this event happens during certain cancer development.

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To sum up, current progress in lncRNAs suggest that lncRNAs are subject to fine-tuned regulation,

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ranging from epigenetic to posttranscriptional regulation, while lncRNAs themselves in turn regulate the whole process, forming a complicated network (Fig 2, left panel). With the integration of lncRNAs, the aberrant network responsible for cancer initiation and development becomes clearer and more

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accurate.

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3.2 LncRNAs in cancer initiation and progression

Cancer initiation and progression is a result of the intricate interaction between the cancer cell and the

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microenvironment. Through the interaction, cancer cells acquire the six hallmarks: sustaining

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proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative

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immortality, inducing angiogenesis, and activating invasion and metastasis[77]. With these hallmarks, cancer obtains the capacities of initiation, growth and metastasis. Previous studies have identified lots

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of the protein-coding genes involved in the process. But the network governing cancer metastasis is far from completion. As how these genes are deregulated and how they confer the cancer cells the capacity are still poorly understood. With the evolving understanding on lncRNAs, accumulating data suggest that lncRNAs are involved in the process and might serve as a hub, moving our understanding on cancer much deeper. 3.2.1 LncRNAs and cancer initiation Although the existence of cancer stem cell and the origin of cancer stem cell are still on debate. It is obviously that there are a subpopulation of the heterogeneous cancer cells responsible for cancer initiation and expansion of the metastasized cancer cells[78]. 16

ACCEPTED MANUSCRIPT To some extent, cancer cell stemness shows similarity with that in the embryonic or adult stem cell. The key factors governing the embryonic and adult stem cells are also involved in the cancer stem

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cell[79]. Recent studies in mouse ES cells suggest that lncRNAs are integral members of control

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machinery of stemness[80], suggesting that lncRNAs might confer cancer cells stemness. In fact, the roles of lncRNAs in cancer cell stemness are now undergoing study and accumulating evidence support the idea that lncRNAs are important in cancer cell stemness acquirement and maintenance, as seen

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from both the stemness related signals regulating lncRNAs and stemness associated downstream targets

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regulated by lncRNAs. Notch signaling is a key developmental pathway that is subject to frequent genetic and epigenetic perturbations in many different human tumors and is considered to be one of the

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key molecules governing the leukemia stem cell in addition to in human T cell acute lymphoblastic

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leukemia (T-ALL). In addition to the canonical mRNAs, a set of T-ALL-specific lncRNA genes are

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directly controlled by the Notch1/Rpbjκ activator complex. And one specific Notch-regulated lncRNA, LUNAR1, is required for efficient T-ALL growth in vitro and in vivo due to its ability to enhance

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IGF1R mRNA expression and sustain IGF1 signaling[81], suggesting an essential role of lncRNAs in leukemia initiation and the stemness. There is increasing evidence that Polycomb repressive complex-1 (PRC1) and -2 (PRC2) play roles in tumor progression and development by blocking differentiation and promoting stem cell self-renewal[82]. The core PRC1 complex includes BMI1, mPh1/2, Pc/Chromobox (CBX), and the ubiquitin E3 ligase RING1A/B, and the PRC2 complex is mainly composed of EED, SUZ12, and the histone lysine methyltransferases EZH1/2. Both BMI1 and EZH2 have been identified to be the key factors for cancer stem cells[82], further suggesting the role of PRC complex in cancer stemness. Recently, PRC complex have been found to interact with many lncRNAs[83], and some of these 17

ACCEPTED MANUSCRIPT lincRNAs are supposed to guide chromatin-modifying complexes to specific genomic loci for gene expression regulation, implicating that lncRNAs are involved in the cancer stemness determination. A

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good example is ANRIL. PRC1 and PRC2 interact with lncRNA ANRIL to form heterochromatin

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surrounding the INK4b-ARF-INK4a locus, leading to its repression. This mechanism provides an increased advantage for bypassing senescence, endowing the cancer cell stemness[84, 85]. Besides ANRIL, oncogenic HOTAIR is also found to interact with PRC complex and relocalize the PRC

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complex towards a metastasis expression profile[86], while metastasis itself is one key trait of cancer

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stem cell[87].

Besides PRC complex, lncRNAs are also found to regulate or regulated by other stemness controllers.

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Multiple lncRNAs are found to be regulated by p53, Oct4, NFkappaB, and Nanog[13, 88], while all of

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these factors are found to be essential for eradication[89] or establishment of the cancer stemness[90].

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LncRNAs are not only the downstream targets of p53, but also the mediators of the p53 function. Both linc-p21 and MEG3 are mediators of p53 function[16, 66].

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Despite these insights, a comprehensive understanding of the roles of lncRNAs in the acquiring and maintaining the stemness or the ability to initiate cancer awaits functional analyses with serial xenograft and other cancer stemness assays. 3.2.2 LncRNAs and cancer growth Cancer growth is the whole process how tumor initiating cells evolve into a visible tumor mass, which referring to cancer cell proliferation, resistance to cell death, and angiogenesis. Numerous studies have demonstrated that cancer related lncRNA changes could promote cancer growth in all of these aspects.Cancer cells enable themselves to proliferate mainly by acquiring the pro-growth signals and evading the growth suppressive signals. Up to now, there are multiple lncRNAs are found to promote 18

ACCEPTED MANUSCRIPT or inhibit cell proliferation, and their aberrant expression contribute to cancer cell growth. PCAT-1, which can promote prostate cancer cell proliferation, is found to be highly upregulated in a subset of

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aggressive prostate cancers [91]. H9 could promote the anchorage-independent growth[73, 74].

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PCGEM1 is another example promoting cell proliferation, whose expression was found to increase in prostate cancers[92-94]. In contrast, GAS5 which can induce cell growth arrest is found to be downregulated in breast cancers[46, 95]. These lncRNAs are just examples in cell proliferation control

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and the emerging lncRNAs are shedding light on the role of lncRNA in cancer cell growth. It is

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important to keep in mind these clarified lncRNAs in cancer cell proliferation are just a small portion of the real case. A recent study identified 216 putative lncRNAs derived from promoter regions of cell

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cycle genes[47]. Some of which, such as lincRNA-p21and ncCCND1, have displayed potent effects on

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altered in human cancers.

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cell cycle control and cell proliferation[37, 42]. It is thus interesting to test whether these transcripts are

Cancer cells should be more prone to death if the cell death machinery is intact. To maintain the

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immortality, cancer cells have evolved to resist cell death by many mechanisms. Besides the above growth arrest role of GAS5 and PCGEM, both of them could also induce apoptosis[92-95]. These novel apoptosis regulators might supply an alternative targets for drug resistance. Most primary solid tumors probably go through a prolonged state of avascular, and angiogenesis starts when hypoxia occurs. Angiogenesis is essential for the tumor to become a visible tumor mass[96]. LncRNAs are also involved in angiogenesis. MALAT1 is found increased in colon, lung and liver cancers[97-99].

Silencing of MALAT1 tips the balance from a proliferative to a migratory endothelial

cell phenotype in vitro, and its genetic deletion or pharmacological inhibition reduces vascular growth in vivo[100], suggesting that MATAT1 could promote cancer by promoting angiogenesis. In contrast, 19

ACCEPTED MANUSCRIPT tumor suppressive MEG3 could inhibit angiogenesis in an in vivo assay[101] and LOC285194 could inhibit the VEGFR1[49].

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3.2.3 LncRNAs and cancer metastasis

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Cancer metastasis is the key reason for cancer death[102]. During the past decades, the whole process how a cancer cell metastasizes to the targeted region is intensively studied, and dozens of famous genes, mainly protein coding genes have been identified and clarified. HOTAIR is significantly increased in

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approximately one-quarter of breast cancer patients, whose expression is strongly predictive of

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eventual metastasis and death. HOTAIR overexpression leads to breast cancer metastasis as seen in an in vivo assay, by changing the cell expression profile favoring metastasis[86].

Besides breast cancer,

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HOTAIR was also observed to be aberrantly expressed in colon, liver, and pancreatic cancers and

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contributes to their invasiveness[103-106]. Accumulating metastasis related lncRNAs are being found.

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Very recently, lncRNA-activated by TGF-β (lncRNA-ATB) was found to promote the invasion-metastasis cascade through multiple mechanisms. On one hand, lncRNA-ATB could

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upregulate ZEB1 and ZEB2 by competitively binding the miR-200 family and then induced EMT and invasion. On the other hand, lncRNA-ATB promoted organ colonization of disseminated tumor cells by binding IL-11 mRNA, autocrine induction of IL-11, and triggering STAT3 signaling[67]. As to how these lncRNAs regulates cancer growth, a common mechanism is that these lncRNAs alters a specific or a set of protein coding genes by acting as decoy, guide, scaffold, or in combinations (For some details, see Table 1 and 2), which in turn alter the expression of lncRNAs, forming a network. Although all of these studies add more details to how the protein coding oncogenes and tumor suppressive genes are deregulated, the lncRNAs in cancer biology is just in its infancy. There remain some fundamental questions to be answered, such as when do the lncRNAs abberantly expressed 20

ACCEPTED MANUSCRIPT regarding the cancer status and stage? Is there a causal role of certain lncRNA aberration in genome instability, as a potent role of lncRNAs in chromatin control? Are there any protein independent

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functions of lncRNAs in cancer biology?

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Expression in patients or cancer cells Overexpressed in renal, breast cancers[68-72]

ANRIL

9p21.3

ncCCND1

11q13

Antisense NcRNA in the INK4 Locus (CDKN2B antisense RNA 1） NA

Inversely relates to p15 expression in leukemia[107], elevated levels in prostate cancer tissues[108] Induced by p53 upon DNA damage[42]

DD3(PCA3)

9q21.22

GAS5

1q25.1

H19

11p15.5

prostate cancer gene 3 noncoding RNA growth-arrest-specif ic transcript 5 H19, imprinted maternally expressed transcript

HOX transcript antisense RNA

increased in primary breast tumours and metastases[86], increased in HCC, GIST, pancreatic cancers[103-106] Increased in esophageal squamous cell carcinoma[59] Increased in HCC and colorectal cancer liver metastasis[33, 58, 75, 111, 112]

HOTAIR

HULC

12q13.13

6p24.3

highly up-regulated in liver cancer

Mechanism deregulation Upregulated prolonged hypoxia NA

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Description of the lncRNA HIF1A antisense RNA 2

CR

aHIF

Genomic location 14q23.2

of

by

Function tumorigenesis biomarker

in

Functional Mechanism NA

Inhibits the expression of p15 and thus cell proliferation (senescence)[84]

NA

Tumor suppressor

Increased in prostate cancer patients[109, 110] Down-regulated in breast cancer[95]

NA

Biomarker

induces TLS allosteric change and silencing cyclin D1 gene expression[42] unknown

NA

Tumor suppressor

Titrates away GR, and thus induces apoptosis and growth arrest[46, 95]

Highly expressed in HCC[73], Correlates with c-Myc in primary breast and lung carcinomas[74]

Upregulated hypoxia[73], Upregulated Myc[74]

Oncogene

H19 knockdown significantly abrogates anchorage-independent growth[73, 74]

NA

Oncogene

increased cancer invasiveness and metastasis in a manner dependent on PRC2 [86], Promotes cell growth[103]

Oncogene/biomarker

Promotes cell proliferation[75]

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Oncogene

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LncRNA

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Table 2 LncRNAs involved in cancer

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by by

SNP Upregulated by HBx[75] and CREB[33], Cancer related SNP [58]

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Increased expression colorectal cancer[65] NA

Upregulated p53[16]

3q13.31

noncoding RNAs LOC285194

Decreased in osteosarcoma[49]

LOH[49]

LOC554202

9p21.3

lncRNA LOC554202

Decreased in triple negative breast cancer[29]

LSINCT5

5p15.33

MALAT1

11q13.1

overexpressed in breast and ovarian cancer[113] Increased in colon, lung and liver cancers[97-99]

MEG3

14q32.2

LincRNA LSINCT5 MALAT1 metastasis associated lung adenocarcinoma transcript 1 Maternally expressed gene 3

ncRAN

17q25.1

Increased in neuroblastoma[52]

aggressive

PCGEM1

2q32.2

Increased in cancer[92-94]

prostate

PCAT-1

8q24

PRNCR1

8q24.2

non-coding RNA expressed in aggressive neuroblastoma Prostate cancer gene expression marker 1 prostate cancer associated transcript 1 prostate cancer

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CR

by

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LOC285194

Imprinting defects[50, 63-65]

Down-regulated cancers[66].

in

NA

NA

Tumor suppressor

Mediates p53-dependent transcriptional repression [16], Inhibits JunB and CTNNB1[37] regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1[49] Inhibits invasiveness through producing miR-31[29]

multiple

Increased in a subset of prostate cancers[91] Increased in prostate cancer[57] 23

Tumor suppressor

Promoter hypermethylation[ 29] NA

Tumor suppressor

oncogene

enhances cellular proliferation[113]

NA

oncogene

Promotes cell migration[97-99]

Aberrant CpG methylation and gene copy number loss[66] Regulated by miR29[76] gain of chromosome 17q

Tumor suppressor

Mediates the effect of p53[66] Inhibits angiogenesis[101]

Oncogene

Induces cell transformation[52]

NA

oncogene

Promotes cell growth and inhibits apoptosis[92-94]

DNA methylation and histone modification[91] SNP[57]

oncogene

Promotes proliferation[91]

oncogene

Promotes cancer cell survival[57]

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6p21.2

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linc-p21

in

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KCNQ1 opposite strand/antisense transcript 1 linRNA-p21

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11p15

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KCNQ1OT1

12q13.12 -q13.13

UC.73a

2q22.3

UCA1

19p13.12

lncRNA-HEIH

5q35

lncRNA-MVIH

10q22

Tumor suppressor[56]

Affect the genes involved in DNA replication, recombination and repair, cellular movement, tumor morphology, and cell death[56]

Tumor suppressor

Titrate the PTEN[35]

Biomarker

Increased expression and altered structure contribute to increased expression of EGLN2[114].

NA

oncogene

Promotes anchorage-dependent and anchorage-independent growth of HCC cells[115] Reduces cancer cell colo320 apoptosis[116] Promotes cell growth[118-120]

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UC.338

Cancer associated SNP destroyed the response to C/EBPα and C/EBPβ[56] Deletion in Melanoma[35, 51] NA

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19q13.2

thyroid

CR

RERT-lncRNA

in

phosphatase and tensin homolog pseudogene 1 long non-coding RNA overlaps with RAB4B and EGLN2 Ultraconserved region 338

Decreased in cancer[35]

Ultraconserved region 73a urothelial carcinoma associated 1 lncRNA High Expression In HCC lncRNA associated with Micro-Vascular Invasion in HCC

high CRC versus normal[116]

NA

Oncogene

Increased in bladder cancer[117]

NA

Oncogene/biomarker

Sp1 mediated transcription[121] NA

oncogene[37]

Increased expression in population of higher risk of hepatocelluar carcinoma[77]

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9p13.3

Down-regulated tumor tissue[56]

increased in human HCC[115]

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PTENP1

non-coding RNA 1 Papillary Thyroid Carcinoma Susceptibility Candidate 3

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14q13.3

Increased in the HBV-related HCC[121] Upregulated in HCC[122]

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PTCSC3

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Note: NA, data not available.

24

Oncogene

miRNAs

targeting

Interacts with EZH2 and promotes cell proliferation[121] activate tumor-inducing angiogenesis[122]

ACCEPTED MANUSCRIPT Taken together, these examples illustrate that misexpressed lncRNAs confer the cancer cell multiple capacities in selfrenewal, growth and metastasis, by changing the interaction with the well-known RNA, DNA and proteins (Fig 2). All of these reported studies are simply the tip of the iceberg. With the

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evolvement of lncRNA in cancer biology, we could see a clearer picture how specific lncRNA confers

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different types of cancer the detailed hallmarks.

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ACCEPTED MANUSCRIPT 4. LncRNAs in cancer diagnosis and therapy Identification and characterization of the detailed lncRNAs involved in the initiation and progression of different types of cancers would be finally beneficial for cancer diagnosis and therapy. Although nearly

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hundreds of oncogenes, tumor suppressor genes and some diagnostic biomarker have been reported in

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the past decades, cancer remains the big hurdle of health. It raises the question whether these protein

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markers and targets really represent the real case of cancer development. And it thus reasonable to question whether we are on the right way to search protein targets for cancer diagnosis and therapy. 4.1 LncRNAs as diagnosis markers

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Different from the protein coding genes, the lncRNAs have the following advantages as a diagnostic marker. (1)LncRNAs coordinately regulate the RNA, DNA and protein biogenesis and function, and

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their important biological function sheds light on its role as a preferential target. (2) Similar as the intensively studied miRNAs, lncRNAs can be easily detected from the body fluid by RT-PCR. Although the current studies on the circulating lncRNAs or lncRNAs in body fluid is rare, the existence

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of lncRNA PCA3 in prostate cancer patient urine samples and lncRNA HULC in HCC patient blood

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has shed light on the role of circulating or secretory lncRNAs on diagnosis[112, 123, 124]. Exosomes are nanovesicles secreted into the extracellular environment upon internal vesicle fusion with the

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plasma membrane. The molecular content of exosomes is a fingerprint of the releasing cell type and of its status. For this reason, and because they are released in easily accessible body fluids such as blood

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and urine, they represent a precious biomedical tool. A growing body of evidence suggests that exosomes may be used as biomarkers for the diagnosis and prognosis of malignant tumors[125]. LncRNAs are found to be secreted in the exosomes and can modulate the function of receptor cells. For example, exosome-mediated transfer of long non-coding RNA ROR can modulate chemosensitivity in human hepatocellular cancer[126]. Specifically, certain oncogenic lncRNAs with low expression levels in Hela and MCF7 cells are enriched in secreted exosomes, such as lincRNA-p21, HOTAIR, ncRNA-CCND1[127], further raising the advantage of lncRNAs in cancer diagnosis.(3) LncRNAs show greater tissue specificity compared to protein-coding mRNAs and miRNAs[9], making them attractive in the search of novel diagnostics/prognostics cancer biomarkers in body fluid samples. Several studies have revealed diagnostic value of lncRNAs in different types of cancer. Increased expression of lncRNA HOTAIR was found to be associated with metastasis in breast cancer[86] and colorectal cancer patients[106], and was related to the recurrence of hepatocellular carcinoma[128]. 26

ACCEPTED MANUSCRIPT Serial studies have suggest that PCA3 level in urine of prostate cancer patients is useful in the diagnosis and minimize unnecessary biopsies, displaying its advantages over the routine PSA biomarker[109].

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Currently, most of the studies linking lncRNA expression profile to cancer diagnosis and prognosis are

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done in cancer samples rather than the exosomes in serum or urine. Due to selective loading, the

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expression profile of lncRNAs in cancer cells might not be linearly related with the exosomes. In addition, the exosomes in the body fluid come from multiple organs are considered. Systemic study testing lncRNAs in the exosomes in different cancer types at different stages is really an important,

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striking but labor intensive work. 4.2 LncRNAs as therapeutic targets

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As discussed above, lncRNAs are not only a marker correlating with the status of cancer, but also an essential contributor to the acquired capacity of cancer. Multiple in vitro cellular and in vivo tumor xenograft assays suggest that knockdown of certain oncogenic lncRNAs has led reduced cancer cell

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growth in many types of cancer cells[11, 129]. In this regard, manipulation of the level of lncRNA

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and/or function in cancer cell provides a novel strategy for cancer therapy. Compared with the routine protein targets, lncRNAs have multiple advantages as cancer therapy target.

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First of all, certain lncRNAs are gene specific epigenetic regulators. It is well established that epigenetic aberrations are common events during the whole process of carcinogenesis, and thus a

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therapeutic intervention at the epigenetic level should be of therapeutic preference. In fact, there are some clinically available or candidate cancer drugs working through epigenetic modulation functions, such as azacytidine, decitabine and zebularine[130]. However, there are no drugs could specificity targeting the chromatin epigenetic modification at particular genomic loci. The ability of lncRNAs to recruit the chromatin modification complexes to particular genomic loci provides us such an opportunity. Such an approach could be less side-effects, and even be useful for cancer prevention [131, 132]. To achieve this goal, detailed lncRNA/mRNA/DNA/protein interaction should be determined under cancer context. The recently established method Chromatin Isolation by RNA Purification (ChIRP) , could map the detailed function of different lncRNAs on the different genomic loci[133], and ultimately fuel the study in this field. As discussed above, lncRNA functions as a platform for complicated interaction based on nucleotide sequence. It is thus rational to design a small RNA or DNA oligos or small chemical molecues to alter the structure of lncRNAs. 27

ACCEPTED MANUSCRIPT Secondly, lncRNAs can become directly functional right after delivery. Unlike protein coding genes, lncRNAs do not encode protein, and thus their function would be soon after delivery and the side-effects would be possibly lower. To this end, lncRNAs can be easily manipulated through RNAi or

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other strategies. RNAi-mediated gene specific silencing holds as a promising strategy in the treatment

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of different diseases, including cancer. Early in 2004, direct intraocular siRNA injection for patients

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with blinding choroidal neovascularization (CNV) has been conducted in human clinical trials[134]. Subsequently, other clinical trials have initiated and move the RNAi based therapy onto approaching clinical application [135-137]. Recently, Davis and colleagues conducted the first in-human phase I

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clinical trial involving the systemic administration of siRNA to patients with solid cancers using a targeted, nanoparticle delivery system, with a significant success[138]. All of these suggest a Besides RNAi

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straightforward approach to selectively silence oncogenic lncRNAs in cancer therapy.

to knockdown of lncRNAs, LNA GapmeRs serve as an alternative strategy. GapmeRs are single-stranded oligonucleotides that consist of a DNA stretch flanked by LNA nucleotides.

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Basepairing with the targeted lncRNA in the nucleus induces degradation by an RNAse H-dependent

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mechanism. Pharmacological inhibition of MALAT1 by GapmeRs reduced blood flow recovery and capillary density after hindlimb ischemia [100]. It is thus interesting to test whether GapmeRs targeting

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MALAT1 could reduce tumor growth in vivo. And this method could be also applied to other nuclear localized oncogenic lncRNAs. In combination, the unique characteristics of lncRNAs have shed light

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on the cancer therapy by targeting lncRNAs. Thirdly, lncRNAs may also have certain advantages at the delivery level. As discussed in the diagnosis section, certain lncRNAs are included in the exosomes and they remain functional when uptaken by the receptor cells[126]. This raises the possibility to deliver lncRNAs through exosomes, and such an endogenous mechanism based gene delivery would be much safer. Exosomes are one of the key players in the cross-talk between cancer and its microenvironment, and exosomes could be used as a vehicle to selectively deliver therapeutic nucleic-acid drugs or conventional drugs for tumor therapy[139]. Compared with other drug delivery system, exosome based delivery has multiple advantages. 1) Exosomes from patient themselves are immune privileged. 2) Exosmes have been shown to cross biological barriers, including the blood–brain barrier and the blood vessels in cancer have endothelial gaps that could transmit exosome size particles much more efficiently and thus providing an targeted accumulation of exosomes in cancer. 3) Exosomes can be modified and thus can be directly targeting 28

ACCEPTED MANUSCRIPT cancer cell specifically. Alvarez-Erviti L et al fused exosomal membrane protein Lamp2b to the neuron-specific RVG peptide. These modified exosomes can specifically enter neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene therapy [140]. Similar strategy can be used

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for cancer therapy by fusing exosome membrane protein with cancer specific peptides. 4) both

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hydrophobic compounds and hydrophilic molecules or macromolecules were reported to be introduced

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into exosomes. For example, curcumin was readily internalized into exosomes after mixing the compound with exosomes[141]. And siRNA could be introduced into exosomes using electroporation [140]. Several studies have provided evidence that exosome can deliver exogenous RNA, including

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siRNA[140] and miRNA[142] to target cells and can be functional both in vitro and in vivo, while studies in exosome mediated lncRNAs are still missing. Since lncRNA itself is one of the components

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of exosomes in cancer condition, all of the above referred advantages make exosome one candidate for drug delivery.

Taken together, the function and characteristics of lncRNAs have made them a good candidate for

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cancer therapy. However, lncRNA oriented anti-tumor drugs or biomolecules have not yet been

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developed as therapies, even in the bench side. There remains a long way to go. 4.3 LncRNA and cancer risk

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Over the past 20 years, a serial studies have demonstrated that common genetic variants could confer susceptibility to different types of cancers[143, 144]. Initially, association studies were conducted using

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a handful of annotated genetic variants. Recently, GWAS study has greatly extended our knowledge of genetic loci associated with human disease risk and other phenotypes. The output of these studies is a series of SNPs (GWAS SNPs) correlated with a phenotype, although not necessarily the functional variants. Notably, 88% of associated SNPs are either intronic or intergenic[145], which are largely neglected due to our previous interest in protein coding genes. As discussed before, there are a large amount of lncRNAs are transcribed from these regions, further suggest that the SNP of lncRNA might confers the differential cancer risk and outcome among populations. For example, a previous genome-wide association study of papillary thyroid carcinoma (PTC) pinpointed two independent SNPs (rs944289 and rs965513) located in regions containing no annotated genes (14q13.3 and 9q22.33, respectively). A very recent study clarified that SNP rs944289 predisposes to PTC through a previously uncharacterized, long intergenic noncoding RNA gene (PTCSC3) that has the characteristics of a tumor suppressor[56]. In fact, more and more lncRNA related SNPs are being revealed and characterized. 29

ACCEPTED MANUSCRIPT GWAS analysis revealed that there is SNP at the lncRNA ANRIL, though its relation with cancer is unknown [146].There is also a SNP that occurs both in the 3’UTR of the zinc finger gene ZFAT and also in the promoter of an antisense transcript. The SNP increases the expression of ZFAT not through

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increasing mRNA stability, but by repressing the expression of the antisense transcript Deregulated

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regulators for LncRNA[147]. Another example is the 4-bp insertion/deletion polymorphism

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(rs10680577) within the distal promoter of EGLN2. The deletion allele was significantly correlated with higher expression of both EGLN2 and RERT-lncRNA, which in turn confers the risk of HCC[114].

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In summary, although our understanding of the molecular mechanisms of lncRNA function is just beginning, the features of lncRNAs have made them ideal candidates for cancer prevention, diagnosis

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and therapeutic intervention.

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ACCEPTED MANUSCRIPT 5. Concluding remarks and future direction The six acquired capabilities(sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and

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metastasis)—the hallmarks of cancer—have provided a useful conceptual framework for understanding

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the complex biology of cancer[77]. Elucidation of the molecular networks by which these hallmark

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capabilities are acquired would eventually lead to the victory of the combat against cancer. It is now becoming obvious that these features are mediated not only by cancer specific-internal and/or external signal induced expression profiles of protein-coding mRNA expression, post-translational

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modifications, localization and translation. The lncRNAs, which function importantly in the regulatory processes, ranging from transcriptional, post-transcriptional, epigenetic and nuclear processes, are also

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a big player in the cancer hallmark acquisition.

Frankly, we are only scratching the surface of the real role of lncRNAs in cancer. Just as the boom of miRNAs study in cancer, which has profoundly changed our understanding of cancer development and

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therapy[148], further studies on the expression, SNP and detailed mechanism how the lncRNAs

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involved in cancer would surely lead to a breakthrough of cancer management. Chief among these may be uncovering the genetic bases of heterogeneity of cancer (different causes, different manifestations,

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and different outcomes) among people, which have remained elusive. Indeed, the concept that lncRNAs serve as a platform and adaptor for diverse DNA–protein, DNA–RNA and DNA–DNA

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interactions, while they are not as conservative as the protein encoding genes and miRNAs[149-151], suggests a distinguished role of lncRNAs in cancer, especially in the personal cancer prevention, diagnosis and therapy. As the lncRNA field continues to evolve, a better understanding of lncRNA biogenesis and function in cancer will certainly affect our understanding of cancer development and management. We anticipate that further elucidation of the detailed lncRNAs involved in tumorigenesis and how they confer cancer cell the hallmarks would ultimately pave the way for rational cancer diagnosis and therapy.

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ACCEPTED MANUSCRIPT Acknowledgements This study was funded by National Science Foundation of China, NSFC31100979, NSFC81170149, and NSFC81101050.

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Conflict of interest

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The authors declare that they have no conflict of interest.

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ACCEPTED MANUSCRIPT References

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ACCEPTED MANUSCRIPT Figure legends Figure 1 lncRNAs in epigenetic, transcriptional and posttranscriptional regulation. There are four types of regulatory mechanisms of epigenetic and transcriptional regulation by

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change of the transcriptional factor towards activation. (b) lncRNAs act as decoy by titrating

chromatin-modifying enzymes to target genes, either in cis or in trans to distant target genes. (d) lncRNAs bring together multiple proteins to form ribonucleoprotein complexes, thus acting as a LncRNAs can also modulate mRNA processing at multiple levels[153]. (e) Antisense

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lncRNAs form duplex with the sense pre-mRNA, and the resultant RNA:RNA duplex might recruit

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ADAR enzymes that catalyze adenosine to inosine conversion. (f) LncRNAs could bind the boundary site between intron and exon of the pre-mRNA, and prevent the alternative splicing. In addition, lncRNAs could also associate with splicing factors and affect splicing. (g) LncRNAs may harbor the

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hairpin structure, which can give rise to the pre-miRNA. (h) LncRNAs may harbor the recognition site

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for functional miRNAs, thus titrate the miRNAs from their mRNA targets. In this category, lncRNAs themselves could be the targets of miRNAs. (i) lncRNAs could compete with miRNAs for binding the

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same mRNAs, the competition would block the recognition of miRNAs and thus increase mRNA translation. (j) lncRNAs:mRNA formed double-stranded structure can direct exosome mediated RNA degradation. One example is that lncRNAs containing Alu repeat elements associate with the Alu

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elements in the 3’UTR, and the resultant double-stranded structure can direct Staufen-mediated decay. (k) lncRNAs bound with the mRNA would positively or negatively modulate the translation efficiency, depending on the mRNA and lncRNA structures.

Figure 2 Dysregulation of lncRNAs in cancer cells alters gene expression by a variety of mechanisms. During the initiation and progression of cancer, the oncogenic and tumor suppressive lncRNAs are deregulated due to epigenetic and genetic aberrations. These altered expression of lncRNAs function as decoy, scaffold, and/or guide for specific regulatory modules, resulting in an gene expression profile in favor of cancer development. The complicated interaction and regulation form a viscous cycle, conferring the cancer cell abilities to self-renew, grow and metastasize. 41

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LncRNAs function as a platform for the complicated interaction with miRNA, mRNA, protein or their combination. LncRNAs have emerged as an essential regulator in almost every aspect of biology. Misexpression of lncRNAs confers the cancer cell capacities for tumor initiation, growth, and metastasis. LncRNAs serve as a promising target for cancer diagnosis and therapy.

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