- Email: [email protected]
Circular RNAs and cancer
Accepted Manuscript Circular RNAs and Cancer Jun He, Qichao Xie, Hailin Xu, Jiantian Li, Yongsheng Li PII:
S0304-3835(17)30201-X
DOI:
10.1016/j.canlet.2017.03.027
Reference:
CAN 13291
To appear in:
Cancer Letters
Received Date: 15 February 2017 Revised Date:
15 March 2017
Accepted Date: 15 March 2017
Please cite this article as: J. He, Q. Xie, H. Xu, J. Li, Y. Li, Circular RNAs and Cancer, Cancer Letters (2017), doi: 10.1016/j.canlet.2017.03.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Circular RNAs and Cancer
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Jun He1,#,*, Qichao Xie2,#, Hailin Xu1, Jiantian Li1, Yongsheng Li2,*
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1 Department of General Surgery, Jiande Branch of The Second Affiliated Hospital,
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School of Medicine, Zhejiang University, Jiande, Zhejiang Province 311600, China.
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2 Institute of Cancer, Xinqiao Hospital, Third Military Medical University,
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Chongqing 400037, China.
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#J.H. and Q.X. contributed equally to this manuscript and share the first authorship. *Correspondence: [email protected] (J. He); [email protected] (Y. Li).
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Word count of the text: 3519.
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Abstract
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Circular RNAs (circRNAs) are a type of non-coding RNA molecules that lack a
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5’-terminal cap and 3’-terminal poly A tail. A large number of circRNAs have been
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identified
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high-throughput sequencing. CircRNA sequence composition determines if a given
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circRNA is exonic, intronic or retained-intronic. CircRNAs are more abundant and
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stable than linear mRNAs, and their expression is both step- and location-specific.
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CircRNAs mediate transcriptional and post-transcriptional regulation of gene and
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protein expression. CircRNAs regulate cancer development via multiple mechanisms,
biological
experiments,
computational
methods
and
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including
sponges,
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epithelial-mesenchymal transition. An in-depth study of circRNA will provide a better
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understanding of carcinogenesis and assist in developing clinical diagnostic and
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therapeutic strategies.
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regulation
of
Wnt
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Keywords: circRNA; cancer; miRNA sponge; transcription.
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signaling
and
the
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1 Introduction Circular RNAs (circRNAs) are a new class of long non-coding RNA which do
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not have 5’ or 3’ ends but are covalently linked to form a closed circular structure.
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Their presence was firstly observed by Sanger in a virus using electron microscopy
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more than 40 years ago [1]. Later, circRNAs in humans, mice, fungi and other
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organisms were subsequently found [2-4]. Due to the structure specificity and low
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abundance, circRNAs were only identified in a few individual genes, including ETS-1
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[5], Sex-determining region Y (SRY) [6], cytochrome P450 2C24 and 2C18 [7, 8] and
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cANRIL (circular ANRIL) [9] over subsequent decades. They were considered as an
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ancient and conserved class of molecules and are the abnormal splicing products of
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RNAs, or the “dark matter” in organisms [10].
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In recent years, molecular purification methods combined with high-throughput
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sequencing technology and improvement of statistical calculation [11] have led to an
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in-depth understanding of these “dark matter”. Specific algorithms and methods for
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use in circRNA research include RNA-seq [12, 13], ssRNA-seq [14], MapSplice [15],
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CircSeq [12], circBase [16], PFOR2 [17] CIRI [18] and segemehl [19]. Using the
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above methods, more than 20,000 circRNAs have been predicted, but they need to be
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verified [12, 20]. Recently, Arraystar pioneered the development of the world’s first
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commercial circRNA chip, which provides an experimental platform for
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systematically characterizing the expression of circRNA in different physiological and
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pathological conditions [21]. Consequently, the development of circRNA in research
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lays a foundation of further exploration of their generation and biofunction in cancer.
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2 Mechanisms of circRNA maturation CircRNAs can be divided into the following three categories based on their
genomic origin and sequence composition (Figure 1).
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2.1 Exonic circRNAs
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Exonic circRNAs are the most abundant circRNAs. Most exonic circRNAs are
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generated from coding genes but do not encode proteins [12, 22, 23]. Generally, 3
ACCEPTED MANUSCRIPT non-coding introns in eukaryotic genes are removed by alternative splicing of
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pre-mRNAs after transcription, followed by sequential linking of exons containing
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protein-coding information to form mature linear RNAs and corresponding protein
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products, this process is called sequential splicing. However, the exonic circRNAs are
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formed by back-splicing, i.e. exon sequences of genes are linked reverse end-to-end.
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There are currently two potential models of back-splicing [12]: lariat-driven
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circularization and intron-pairing-driven circularization. The former suggests that
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during transcription of pre-RNA, partial splicing of the RNA (i.e., covalent binding of
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the splice donor of one exon to the splice acceptor of a different exon narrows the
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distances between the original non-adjacent exons) leads to exon skipping and the
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formation of a circular RNA intermediate. Subsequently, exonic circRNA is formed
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by intra-lariat splicing.
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The intron-pairing-driven circularization model has been extensively validated.
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This model indicates that exons involved in circularization are connected to introns
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containing reverse complementation sequences that result in spatially close donor and
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acceptor splice exons that form circRNAs. In the 1990s, it was discovered that
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circularization of circEts-1 and circSry driven by complementary sequence pairing [5].
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It should be noted, however, that not all circRNAs are generated by complementary
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regions on either side of an exon. For example, Wang et al. constructed a minigene in
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vitro and found that the reverse complementation of exon-flanking introns upstream
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and downstream is not necessary for the formation of circRNAs [24]. In 2014,
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Ashwal-Fluss et al. identified an antagonistic relationship between linear mRNA
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splicing and back splicing of circRNAs [25]. Pre-mRNAs containing stable 3’ ends
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were more likely to form circRNAs [26], suggesting that circRNAs could be produced
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after transcription rather than during co-transcription, as has been described for
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eukaryotic pre-mRNAs.
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One common feature was noted in both of the models described, i.e., long
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flanking introns of complementary ALU repeats [12]. The exon circularization is
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mediated by intron-pairing introns [27]. Human genome intron regions contain a large
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number of complementary sequences whose pairing can also produce multiple 4
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circRNAs through a mechanism called alternative circularization.
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2.2 CiRNAs Introns account for more than 20% of the human genome, where most introns
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form a lasso structure after splicing that is degraded after off-branch [28]. However,
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some introns containing certain key nucleic acid sequences cannot debranch after
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splicing and instead form intron-derived ciRNAs. It is known that the production of
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ciRNAs is dependent on conserved motifs at both ends of the intron, i.e., the
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7-nucleotide GU-rich motif located at the 5’ splicing site and the 11-nucleotide C-rich
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motif at 3’-branch site. These nucleotides prevent debranching and cause the RNA to
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retain a circular structure [27]. Potential additional protein factors involved in the
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formation of ciRNAs are unknown. The distinct method of formation of ciRNAs
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results in distinct differences from exonic circRNAs: ciRNAs are 2’-5’
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phospholipid-linked molecules, and exonic circRNAs are 3’-5’ phospholipid-linked
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molecules. CiRNAs are present in the nucleus and are able to bind to and regulate the
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expression of parent genes.
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2.3 Retained-intron circRNAs
In the process of back-splicing to form exonic circRNAs, circular RNAs
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containing un-spliced introns can stabilize and exist [20], potentially acting as an
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intermediate in the splicing process, or to form distinct circRNAs. By preparing RNA
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polymerase (Pol)
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immunoprecipitating with Pol II antibody) followed by RNA sequencing and
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bioinformatics analysis, Li et al. found more than 100 circRNAs interacting with Pol
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II in human cells [29]. Further studies have shown that these circular RNAs are also
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formed by exon back-splicing, but this type of circRNAs contain both exons and
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introns and are fully localized to the nucleus. Hence, these specific circRNAs were
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termed exon-intron circRNAs (EIciRNAs).
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3 CircRNA biofunctions 5
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RNA and
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3.1 As a ceRNA or miRNA sponge Competitive endogenous RNA (ceRNA) contains a miRNA response element
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(MRE), which competitively binds miRNA [30, 31]. Therefore, ceRNA can affect the
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regulatory functions of miRNAs in gene expression and reduce the inhibitory effect of
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miRNAs on target molecules. Recent studies have shown that exonic circRNAs can
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act as ceRNA or miRNA sponge molecules, which regulate the expression of genes by
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adsorbing miRNAs (Figure 2A) [32]. CDR1as (antisense to the cerebellar
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degeneration-related protein 1 transcript) is a natural antisense transcript (NAT) of the
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cerebellar degeneration-associated protein 1 (CDR1) gene. This circRNA is
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approximately 1.5 kb in length and contains 74 binding sites for miR-7. More than 60
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sites are conserved and are capable of binding to RNA-induced silencing complexes
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(RISC) formed by miR-7 and Ago2 protein [33]. Therefore, CDR1as is also called
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ciRS-7 (circular RNA sponge for miR-7). The circRNA CDR1as co-localizes with
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miR-7 in the cytoplasm. Knockdown of CDR1as or overexpression of miR-7
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promotes the degradation of miR-7-target mRNAs. Conversely, overexpression of
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CDR1as inhibits the action of miR-7 and can mimic the phenotype of morpholino
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miR-7 knockdown, leading to a decrease of the midbrain size in embryos of zebrafish
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[13]. Hansen et al. found that although CDR1as cannot be degraded by
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miR-7-mediated RISC, it can complement miR-671 and degrade [34], suggesting that
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miR-671 indirectly regulates miR-7 by lowering CDR1as.
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SRY gene consists of only one exon. In the early stages of development, its
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transcript forms a linear RNA, serving as a template for protein synthesis. However,
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in adult testes, its cytoplasmic RNA is mainly circular and does not undergo
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translation [6]. Studies have confirmed that the reverse repeat sequence on both sides
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of the SRY exon can be directly transcribed into a circRNA molecule [35, 36]. The
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circular transcript of SRY gene has a similar function to CDR1as, in that it contains 16
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MREs of miRNA-138 and displays as the miR-138 sponge, thereby regulating the
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expression of miR-138-target genes [37]. In addition, Li et al. found that circ-ITCH
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could be used as a sponge of miR-7, miR-17 and miR-214 [38]. Bioinformatics
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analysis has shown that tens of thousands of circular RNAs have miRNA adsorption
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functional capabilities [39], but few have been validated. Therefore, universal
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circRNA sponge function and regulation of miRNAs and ceRNAs requires
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elucidation.
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3.2 Regulating gene transcription
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Recent studies have shown that circRNA can regulate parental gene expression.
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For example, The formation of circRNAs is dependent on key flanking RNA elements
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that are critical for intron lasso-circularization eluting debranching [27]. These
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circRNAs do not enrich miRNA targets, indicating that their functions are unique.
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CircRNAs that regulate the transcription of parental genes include some ciRNAs (e.g.,
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ci-ankrd52, ci-sirt7) and EIciRNAs (e.g., circEIF3J, circPAIP2).
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Detailed DNA/RNA duplex in situ hybridization showed that some ciRNAs are
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localized to their transcriptional sites and can bind RNA pol II complexes to affect the
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transcription of RNA pol II via an unknown mechanism, thereby cis-regulating their
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parent genes (Figure 2B). Interestingly, ciRNAs not only are enriched at their
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transcriptional sites but also accumulate in other regions of the nucleus, suggesting
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that they may play a trans-regulatory role. Ci-ankrd52 is an ankrd52-derived ciRNA
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that specifically binds RNA pol II actively transcribed by its parent gene ankrd52 to
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regulate the transcription efficiency of ankrd52. Upon ci-ankrd52 knock out, ankrd52
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transcription efficiency is significantly reduced, however, administration of high
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levels of ci-ankrd did not improve the transcription efficiency of ankrd52.
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Researchers speculate that this may be caused by the abnormal positioning of
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exogenous ci-ankrd52 [27]. Silent information regulator 7 (ci-sirt7) also acts via a
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similar mechanism [27]. In addition, cANRIL, an exonic circRNA, is also presumed
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have transcriptional regulation activity. ANRIL inhibits transcription of its encoding
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gene INK4/ARF by binding to the Polycomb Gene (PcG) complex. ANRIL transcripts
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can undergo back-splicing to form cANRIL, suggesting that formation of cANRIL
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reduces ANRIL, thus regulating transcription of INK4/ARF [9].
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Studies of two EIciRNAs, namely, EIciEIF3J and EIciPAIP2, show that both
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co-localize with their parental locus and U1 snRNP (small nuclear ribonucleoprotein), 7
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interaction of RNA pol II with the promoter region of the parental gene resulting in in
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cis transcription initiation of the parent gene. Consistently, 3’ untranslated regions
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(3’-UTR) of cir-ITCH and ITCH have been found to share miRNA binding sites [29].
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Further studies have demonstrated that cir-ITCH interacts with miR-7, miR-17 and
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miR-214 to up-regulate ITCH expression [38]. It is speculated that exon-derived
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circRNAs play a regulatory role in the cytoplasm, while intron-derived circRNAs
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(such as ciRNA and EIciRNA) play a transcriptional regulatory role in the nucleus.
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3.3 Regulating RNA binding proteins
The adsorption of protein factors by linear long non-coding RNA (lncRNA) has
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been reported. For example, sno-lncRNAs regulate alternative splicing of downstream
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genes by adsorbing the alternative splicing factor Fox2 [40]. CircRNA molecules can
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also adsorb protein factors. RNA splicing factor MBL can bind exon 2 of its parental
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gene and promote its circularization to form circMBL. CircMBL can then bind MBL,
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reducing the effective concentration of MBL and the production of circMBL [25]. The
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binding of intron-exon circular RNA circEIF3J with U1 RNA promotes the binding of
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U1 snRNP complex and RNA pol II and enhances parental gene transcription (Figure
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2C) [29]. The complex formation of circ-Foxo3, kinase inhibitor protein (p21) and
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cell division protein kinase 2 (CDK2) inhibits the promotion of CDK2 on cell division
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and blunts cell cycle progression [41]. CDR1as and circSry can bind with the miRNA
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effector AGO to cleave it, inhibit its translation, and eventually promotes its
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degradation (Figure 2C) [13, 33]. Ci-ankrd52 can interact with the RNA pol II
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complex to affect its activity and ultimately regulate transcription [27]. These findings
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suggest that circRNAs can serve as a scaffolding platform for protein-RNA,
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protein-DNA, and protein-protein interactions.
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3.4 Protein translation
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CircRNAs can also be translated to proteins, similar to linear mRNAs. In 1995,
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Chen and Sarnow et al. found that an internal ribosome entry site (IRES) and the 8
ACCEPTED MANUSCRIPT initiation codon ATG in a specific circRNA would allow the circRNA translation
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template to function as mRNA (Figure 2D) [42]. In 2014, Jeck et al. found that many
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exon-derived circRNAs contain translation initiation sites. CircRNAs regulate protein
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expression by blocking the translation initiation site [43]. Wang et al. constructed a
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minigene in vitro containing the cytomegalovirus (CMV) promoter, IRES, and an
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exon encoding green fluorescent protein (GFP), which allowed the circularization of
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corresponding transcript [24]. After transfection into cells, this minigene transcript
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was able to form circular RNA that was capable of translating GFP protein.
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Interestingly, to date, only one naturally occurring circRNA can encode proteins in
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eukaryotic cells, namely hepatitis D virus (HDV). HDV is a satellite virus of HBV
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(hepatitis B virus). HDV and HBV virus particles co-embedded and produce the same
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protein; however, translation of the protein is not regular, likely due to the viral vector.
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The role of circRNAs in control of translation still warrants further study [44].
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4 CircRNAs in cancer
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Studies have shown a close relationship between circRNAs and a variety of
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tumors, including colon cancer, ovarian cancer, gastric cancer, esophageal cancer, and
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glioma [38, 45-49]. The wide existence, high stability and variety of regulatory
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functions of circRNAs are undoubtedly a novel area of interest in the early diagnosis
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and treatment of cancer. Some clinical studies have shown that the levels of certain
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circRNAs are decreased in tumor tissue in comparison to normal tissue and that
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circRNA levels are significantly correlated with clinical features of distant metastasis,
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staging, age of onset, and gender. CircRNA expression in tumor tissue and tumor cell
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lines were found reduced, and the ratio of specific circRNAs and their linear isomers
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was significantly different in healthy tissue and that affected by colorectal cancer
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when analyzed by transcriptome analysis [50], providing a new molecular focus in the
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study of pathogenesis of tumors. Many tumor-associated chromosomal translocation
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regions produce fusion circRNAs (f-circRNAs) , which might promote cancer growth
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[51]. CircRNAs are also enriched in exosomes [52], indicating that it might be
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possible to diagnose cancer by detecting serum circRNAs.
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4.1 Cancer biology-related circRNA signal pathway CircRNAs are involved in the regulation of the molecular biology of tumors via
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multiple mechanisms. For instance The expression of circ-ITCH in human esophageal
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cancer tissues is lower than that in para-cancerous tissue. Circ-ITCH can inhibit the
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activity of the Wnt pathway in the esophageal cancer cell lines Eca-109 and TE-1,
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resulting in inhibition of the proliferative ability, cell cycle progression and
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tumorigenic ability [38]. In addition, circRNAs also regulate epithelial-mesenchymal
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transition (EMT). During the process of EMT, QUAKING protein can bind to the
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flanking sequence of specific circRNAs and regulate the production of these
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circRNAs. Of note, the number of such circRNA has reached thousands, suggesting
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an inextricable link between circRNAs and cancer development [53].
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4.2. CircRNA and cancer-related miRNA
Hansen originally proposed the idea that circRNAs can target miRNAs [32].
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Recently, circRNAs were shown to play an important role in the regulation of
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miRNA-mediated gene expression by isolation of miRNAs. Subsequently, Ghosal et
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al. performed gene cluster analysis (GO) and analyzed protein-coding loci of
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miRNA-circRNA-related diseases to detect gene enrichment associated with specific
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physiological processes [54]. This was the first comprehensive data analysis of the
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role of circRNA in cancer, however the relationship between circRNA-miRNAs
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requires investigation.
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CDR1as/miR-7 can affect carcinogenesis and progression of disease in a variety
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of ways (Figure 3A) [34]. Human ciRS-7 is highly expressed in cells and interacts
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with
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miRNA-mediated degradation and significantly inhibit miR-7 activity, thereby
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up-regulating miR-7 target gene expression [32]. Owing to the high expression of
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ciRS-7 and miR-7 in brain tissue, the frequent ciRS-7/miR-7 interaction will change
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intracellular levels of RISC. Therefore, miRNAs and miRNA regulatory activity in
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miR-7/ciRS-7 overexpressing tissues are inactive [34].
AGO
protein
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dependent
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CircRNAs
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such as epidermal growth factor receptor (EGFR), insulin receptor substrate-1 (IRS-1),
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activated CDC42 kinase 1 (Ack1), and phosphatidylinositol 3-kinase catalytic subunit
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δ (PIK3CD) (Figure 3B) [55-59]. MiR-7 can effectively inhibit the expression of
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EGFR and reduce the expression of IRS-1 and IRS-2 by inhibiting protein kinase B
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(PKB) resulting in reduced activity and invasion of glioma cells [56]. Pak1 is a
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member of serine threonine protein kinases. Endogenous miR-7 is negatively
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correlated with levels of Pak1 and positively correlated with Homeobox D10
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(HOXD10), a homology domain transcription factor. During the transformation of
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low-invasive to highly invasive breast cancer, expression of Pak1 was up-regulated
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while the expression of miR-7 and HOXD10 were down-regulated. In highly
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aggressive breast cancer cells, miR-7 inhibited the proliferative activity, invasion and
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tumorigenic potential. These results indicate that miR-7/Pak1 pathway plays an
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important role in the development of breast cancer [57]. In lung cancer, breast cancer
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and glioblastoma cell lines, miR-7 significantly reduces the expression of
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EGFR-related mRNAs, including Raf1, protein kinase B/Akt (PKB/Akt) and
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extracellular signal-regulated kinase (ERK) 1/2 [59]. In schwannoma cells, Ack1 is a
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direct target of miR-7, and its expression is inversely proportional to that of miR-7
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[58]. In vitro, miR-7 overexpression is involved in cell cycle arrest and cell migration
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regulated by the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway.
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Additionally the expression of Akt, mammalian target of rapamycin (mTOR) and
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p70S6K are down-regulated in HCC cells [55]. MiR-7 inhibits the growth of human
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non-small cell lung cancer A549 cells by regulating the expression of B-cell
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lymphoma-2 (BCL-2), an apoptosis-related gene. Cancer stem cells (CSC) play an
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important role in cancer progression and metastasis. It has been confirmed that miR-7
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is downregulated compared to all other miRNAs in CSC [60]. Kruppel-like factor 4
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(KLF4) is a key transcription factor in CSC. The anticancer effect of miR-7 may be
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partly due to its concomitant inhibition of KLF4, which plays an important regulatory
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role in metastasis of breast cancer [61]. In addition, miR-7 up-regulates the expression
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of cadherin to indirectly inhibit EMT by suppressing insulin-like growth factor 1
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receptor (IGF1R) and focal adhesion kinase, resulting in delayed tumor growth and
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metastasis. This mechanism is important in the tumorigenesis of gastric cancer,
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squamous cell carcinoma, breast cancer and malignant glioma [62-65]. However, some studies reported a cancer-promoting role of miR-7. For example,
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miR-7 down-regulated compared to all other miRNAs in colorectal cancer. It targets
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oncogene YY1, leading to P53 inactivation [66]. However, the expression of CDR1as
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is significantly increased in colorectal cancer tissues. In the lung cancer CL1-5 cell
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line, miR-7 levels are positively correlated with increased transplanted tumor volume
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and the survival rate of nude mice [67]. In cervical cancer and lung adenocarcinoma
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cell lines, inhibition of miR-7 resulted in decreased proliferation and increased
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apoptosis, suggesting that overexpression of miR-7 may not lead to suppression of
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carcinogenesis [68]. This is also shown in renal cell carcinoma, where invasion and
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proliferation are associated with high expression of miR-7 [69]. Altogether, miR-7 is
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closely related to ciRS-7, and the micro-regulatory axis of miR-7/miR-671/ciRS-7
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may play an important role in cancer pathophysiology.
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4.3 CircRNA and cancer therapy
Use of gene knockout, antisense oligonucleotides and miRNA sponges are three
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classical approaches to cause loss of miRNA function [70]. Construction of knockout
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animal models is time-consuming, expensive and difficult. Chemically modified
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antisense oligonucleotides are useful in short-term experiments, but cannot achieve
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long-term miRNA inhibition. miRNA sponge technology is a new technology and
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possible alternative to gene knockout technology [71]. miRNA sponges transfected
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into human cells have similar miRNA inhibiting potency to antisense oligonucleotides.
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Compared with conventional linear miRNA sponges containing a single MRE,
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circRNA sponges contain several MREs. In malignant melanoma cell lines, circRNA
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sponges exhibited superior anticancer effects when compared to linear sponges.
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Circular sponges may be the best intracellular means of inhibiting miRNA activity.
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Importantly, these sponges could be used to target oncogenes and reverse the
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malignant phenotype of human cancer cells [72]. Artificial sponges are a powerful
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Liu et al. constructed a circRNA sponge targeting miR-21 and miR-221 and used it to
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treat melanoma cell lines. The results demonstrate that the circRNA sponge showed
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more potent anti-cancer effect than linear RNA [73]. These findings suggest that
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synthetic circRNA inhibitors are a novel approach for future cancer therapies.
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5 Concluding remarks
With improvements in the new generation of high-throughput sequencing and
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biological technologies, different researchers continue to gain an in-depth
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understanding of circRNAs. These molecules act as an miRNA molecule sponge,
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interact with RNA-binding proteins, and participate in gene transcriptional regulation.
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However, there are many other unknown functional mechanisms to be investigated.
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The identification and functional study of these new RNA molecules not only enrich
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our understanding of the complexity of the eukaryotic transcriptome and non-coding
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RNA but also provide new ideas and methods for the diagnosis and treatment of
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human diseases. We can appropriately modify circular RNA molecules to silence
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important binding sites associated with cancer. We can also target specific molecular
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drugs to change downstream gene expression in order to treat cancer. At the same
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time, emerging and improved methods of artificially constructing circular RNAs or
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interfering RNAs makes it possible to regulate the expression of intracellular
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circRNAs, which is essential to further explore the function of circRNAs [13, 53].
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The use of circRNAs in clinical diagnosis and treatment has become a new foothold
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for translational and precision medicine. The appropriate and precise use referring to
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circRNAs and understanding their functionalities and mechanisms are the key topics
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in the field of circRNAs and cancer research in the years to come. The continuous
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exploration and research in this field will provide an important molecular basis for
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understanding the complex regulation of life activities.
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ACCEPTED MANUSCRIPT Conflicts of Interest: The authors have declared no conflicts of interest
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Acknowledgements: The work was supported by Youth 1000 Talent Plan and the
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National Natural Science Foundation of China (81472435 and 81671573).
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[1] H.L. Sanger, G. Klotz, D. Riesner, H.J. Gross, A.K. Kleinschmidt, Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures, Proc Natl Acad Sci U S A, 73 (1976) 3852-3856. [2] D. Kolakofsky, Isolation and characterization of Sendai virus DI-RNAs, Cell, 8 (1976) 547-555. [3] Y. Matsumoto, R. Fishel, R.B. Wickner, Circular single-stranded RNA replicon in Saccharomyces cerevisiae, Proc Natl Acad Sci U S A, 87 (1990) 7628-7632. [4] J.M. Nigro, K.R. Cho, E.R. Fearon, S.E. Kern, J.M. Ruppert, J.D. Oliner, K.W. Kinzler, B. Vogelstein, Scrambled exons, Cell, 64 (1991) 607-613. [5] C. Cocquerelle, P. Daubersies, M.A. Majerus, J.P. Kerckaert, B. Bailleul, Splicing with inverted order of exons occurs proximal to large introns, EMBO J, 11 (1992) 1095-1098. [6] B. Capel, A. Swain, S. Nicolis, A. Hacker, M. Walter, P. Koopman, P. Goodfellow, R. Lovell-Badge, Circular transcripts of the testis-determining gene Sry in adult mouse testis, Cell, 73 (1993) 1019-1030. [7] P.G. Zaphiropoulos, Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping, Proc Natl Acad Sci U S A, 93 (1996) 6536-6541. [8] P.G. Zaphiropoulos, Exon skipping and circular RNA formation in transcripts of the human cytochrome P-450 2C18 gene in epidermis and of the rat androgen binding protein gene in testis, Mol Cell Biol, 17 (1997) 2985-2993. [9] C.E. Burd, W.R. Jeck, Y. Liu, H.K. Sanoff, Z. Wang, N.E. Sharpless, Expression of linear and novel circular forms of an INK4/ARF-associated non-coding RNA correlates with atherosclerosis risk, PLoS Genet, 6 (2010) e1001233. [10] C. Cocquerelle, B. Mascrez, D. Hetuin, B. Bailleul, Mis-splicing yields circular RNA molecules, FASEB J, 7 (1993) 155-160. [11] F. Caiment, S. Gaj, S. Claessen, J. Kleinjans, High-throughput data integration of RNA-miRNA-circRNA reveals novel insights into mechanisms of benzo[a]pyrene-induced carcinogenicity, Nucleic Acids Res, 43 (2015) 2525-2534. [12] W.R. Jeck, J.A. Sorrentino, K. Wang, M.K. Slevin, C.E. Burd, J. Liu, W.F. Marzluff, N.E. Sharpless, Circular RNAs are abundant, conserved, and associated with ALU repeats, RNA, 19 (2013) 141-157. [13] S. Memczak, M. Jens, A. Elefsinioti, F. Torti, J. Krueger, A. Rybak, L. Maier, S.D. Mackowiak, L.H. Gregersen, M. Munschauer, A. Loewer, U. Ziebold, M. Landthaler, C. Kocks, F. le Noble, N. Rajewsky, Circular RNAs are a large class of animal RNAs with regulatory potency, Nature, 495 (2013) 333-338. [14] T. Lu, L. Cui, Y. Zhou, C. Zhu, D. Fan, H. Gong, Q. Zhao, C. Zhou, Y. Zhao, D. Lu, J. Luo, Y. Wang, Q. Tian, Q. Feng, T. Huang, B. Han, Transcriptome-wide investigation of circular RNAs in rice, RNA, 21 (2015) 2076-2087. [15] K. Wang, D. Singh, Z. Zeng, S.J. Coleman, Y. Huang, G.L. Savich, X. He, P. Mieczkowski, S.A. Grimm, C.M. Perou, J.N. MacLeod, D.Y. Chiang, J.F. Prins, J. Liu, MapSplice: accurate mapping of RNA-seq reads for splice junction discovery, Nucleic Acids Res, 38 (2010) e178.
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References
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[16] P. Glazar, P. Papavasileiou, N. Rajewsky, circBase: a database for circular RNAs, RNA, 20 (2014) 1666-1670. [17] Z. Zhang, S. Qi, N. Tang, X. Zhang, S. Chen, P. Zhu, L. Ma, J. Cheng, Y. Xu, M. Lu, H. Wang, S.W. Ding, S. Li, Q. Wu, Discovery of replicating circular RNAs by RNA-seq and computational algorithms, PLoS Pathog, 10 (2014) e1004553. [18] Y. Gao, J. Wang, F. Zhao, CIRI: an efficient and unbiased algorithm for de novo circular RNA identification, Genome Biol, 16 (2015) 4. [19] S. Hoffmann, C. Otto, G. Doose, A. Tanzer, D. Langenberger, S. Christ, M. Kunz, L.M. Holdt, D. Teupser, J. Hackermuller, P.F. Stadler, A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection, Genome Biol, 15 (2014) R34. [20] J. Salzman, R.E. Chen, M.N. Olsen, P.L. Wang, P.O. Brown, Cell-type specific features of circular RNA expression, PLoS Genet, 9 (2013) e1003777. [21] X. Fan, X. Zhang, X. Wu, H. Guo, Y. Hu, F. Tang, Y. Huang, Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos, Genome Biol, 16 (2015) 148. [22] J. Salzman, C. Gawad, P.L. Wang, N. Lacayo, P.O. Brown, Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types, PLoS One, 7 (2012) e30733. [23] J.U. Guo, V. Agarwal, H. Guo, D.P. Bartel, Expanded identification and characterization of mammalian circular RNAs, Genome Biol, 15 (2014) 409. [24] Y. Wang, Z. Wang, Efficient backsplicing produces translatable circular mRNAs, RNA, 21 (2015) 172-179. [25] R. Ashwal-Fluss, M. Meyer, N.R. Pamudurti, A. Ivanov, O. Bartok, M. Hanan, N. Evantal, S. Memczak, N. Rajewsky, S. Kadener, circRNA biogenesis competes with pre-mRNA splicing, Mol Cell, 56 (2014) 55-66. [26] D. Liang, J.E. Wilusz, Short intronic repeat sequences facilitate circular RNA production, Genes Dev, 28 (2014) 2233-2247. [27] Y. Zhang, X.O. Zhang, T. Chen, J.F. Xiang, Q.F. Yin, Y.H. Xing, S. Zhu, L. Yang, L.L. Chen, Circular intronic long noncoding RNAs, Mol Cell, 51 (2013) 792-806. [28] F. Rodriguez-Trelles, R. Tarrio, F.J. Ayala, Origins and evolution of spliceosomal introns, Annu Rev Genet, 40 (2006) 47-76. [29] Z. Li, C. Huang, C. Bao, L. Chen, M. Lin, X. Wang, G. Zhong, B. Yu, W. Hu, L. Dai, P. Zhu, Z. Chang, Q. Wu, Y. Zhao, Y. Jia, P. Xu, H. Liu, G. Shan, Exon-intron circular RNAs regulate transcription in the nucleus, Nat Struct Mol Biol, 22 (2015) 256-264. [30] L. Poliseno, L. Salmena, J. Zhang, B. Carver, W.J. Haveman, P.P. Pandolfi, A coding-independent function of gene and pseudogene mRNAs regulates tumour biology, Nature, 465 (2010) 1033-1038. [31] M. Cesana, D. Cacchiarelli, I. Legnini, T. Santini, O. Sthandier, M. Chinappi, A. Tramontano, I. Bozzoni, A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA, Cell, 147 (2011) 358-369. [32] T.B. Hansen, T.I. Jensen, B.H. Clausen, J.B. Bramsen, B. Finsen, C.K. Damgaard, J. Kjems, Natural RNA circles function as efficient microRNA sponges, Nature, 495
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(2013) 384-388. [33] T.B. Hansen, E.D. Wiklund, J.B. Bramsen, S.B. Villadsen, A.L. Statham, S.J. Clark, J. Kjems, miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA, EMBO J, 30 (2011) 4414-4422. [34] T.B. Hansen, J. Kjems, C.K. Damgaard, Circular RNA and miR-7 in cancer, Cancer Res, 73 (2013) 5609-5612. [35] Y.W. Jeske, J. Bowles, A. Greenfield, P. Koopman, Expression of a linear Sry transcript in the mouse genital ridge, Nat Genet, 10 (1995) 480-482. [36] A. Hacker, B. Capel, P. Goodfellow, R. Lovell-Badge, Expression of Sry, the mouse sex determining gene, Development, 121 (1995) 1603-1614. [37] M.W. Hentze, T. Preiss, Circular RNAs: splicing's enigma variations, EMBO J, 32 (2013) 923-925. [38] F. Li, L. Zhang, W. Li, J. Deng, J. Zheng, M. An, J. Lu, Y. Zhou, Circular RNA ITCH has inhibitory effect on ESCC by suppressing the Wnt/beta-catenin pathway, Oncotarget, 6 (2015) 6001-6013. [39] L.F. Thomas, P. Saetrom, Circular RNAs are depleted of polymorphisms at microRNA binding sites, Bioinformatics, 30 (2014) 2243-2246. [40] Q.F. Yin, L. Yang, Y. Zhang, J.F. Xiang, Y.W. Wu, G.G. Carmichael, L.L. Chen, Long noncoding RNAs with snoRNA ends, Mol Cell, 48 (2012) 219-230. [41] W.W. Du, W. Yang, E. Liu, Z. Yang, P. Dhaliwal, B.B. Yang, Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2, Nucleic Acids Res, 44 (2016) 2846-2858. [42] C.Y. Chen, P. Sarnow, Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs, Science, 268 (1995) 415-417. [43] W.R. Jeck, N.E. Sharpless, Detecting and characterizing circular RNAs, Nat Biotechnol, 32 (2014) 453-461. [44] A. Kos, R. Dijkema, A.C. Arnberg, P.H. van der Meide, H. Schellekens, The hepatitis delta (delta) virus possesses a circular RNA, Nature, 323 (1986) 558-560. [45] Y. Dou, D.J. Cha, J.L. Franklin, J.N. Higginbotham, D.K. Jeppesen, A.M. Weaver, N. Prasad, S. Levy, R.J. Coffey, J.G. Patton, B. Zhang, Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes, Sci Rep, 6 (2016) 37982. [46] I. Ahmed, T. Karedath, S.S. Andrews, I.K. Al-Azwani, Y.A. Mohamoud, D. Querleu, A. Rafii, J.A. Malek, Altered expression pattern of circular RNAs in primary and metastatic sites of epithelial ovarian carcinoma, Oncotarget, 7 (2016) 36366-36381. [47] J. Chen, Y. Li, Q. Zheng, C. Bao, J. He, B. Chen, D. Lyu, B. Zheng, Y. Xu, Z. Long, Y. Zhou, H. Zhu, Y. Wang, X. He, Y. Shi, S. Huang, Circular RNA profile identifies circPVT1 as a proliferative factor and prognostic marker in gastric cancer, Cancer Lett, 388 (2016) 208-219. [48] W. Xia, M. Qiu, R. Chen, S. Wang, X. Leng, J. Wang, Y. Xu, J. Hu, G. Dong, P.L. Xu, R. Yin, Circular RNA has_circ_0067934 is upregulated in esophageal squamous cell carcinoma and promoted proliferation, Sci Rep, 6 (2016) 35576. [49] P. Yang, Z. Qiu, Y. Jiang, L. Dong, W. Yang, C. Gu, G. Li, Y. Zhu, Silencing of
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cZNF292 circular RNA suppresses human glioma tube formation via the Wnt/beta-catenin signaling pathway, Oncotarget, 7 (2016) 63449-63455. [50] A. Bachmayr-Heyda, A.T. Reiner, K. Auer, N. Sukhbaatar, S. Aust, T. Bachleitner-Hofmann, I. Mesteri, T.W. Grunt, R. Zeillinger, D. Pils, Correlation of circular RNA abundance with proliferation--exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues, Sci Rep, 5 (2015) 8057. [51] J. Guarnerio, M. Bezzi, J.C. Jeong, S.V. Paffenholz, K. Berry, M.M. Naldini, F. Lo-Coco, Y. Tay, A.H. Beck, P.P. Pandolfi, Oncogenic Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal Translocations, Cell, 165 (2016) 289-302. [52] P. Li, S. Chen, H. Chen, X. Mo, T. Li, Y. Shao, B. Xiao, J. Guo, Using circular RNA as a novel type of biomarker in the screening of gastric cancer, Clin Chim Acta, 444 (2015) 132-136. [53] S.J. Conn, K.A. Pillman, J. Toubia, V.M. Conn, M. Salmanidis, C.A. Phillips, S. Roslan, A.W. Schreiber, P.A. Gregory, G.J. Goodall, The RNA binding protein quaking regulates formation of circRNAs, Cell, 160 (2015) 1125-1134. [54] S. Ghosal, S. Das, R. Sen, P. Basak, J. Chakrabarti, Circ2Traits: a comprehensive database for circular RNA potentially associated with disease and traits, Front Genet, 4 (2013) 283. [55] Y. Fang, J.L. Xue, Q. Shen, J. Chen, L. Tian, MicroRNA-7 inhibits tumor growth and metastasis by targeting the phosphoinositide 3-kinase/Akt pathway in hepatocellular carcinoma, Hepatology, 55 (2012) 1852-1862. [56] B. Kefas, J. Godlewski, L. Comeau, Y. Li, R. Abounader, M. Hawkinson, J. Lee, H. Fine, E.A. Chiocca, S. Lawler, B. Purow, microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma, Cancer Res, 68 (2008) 3566-3572. [57] S.D. Reddy, K. Ohshiro, S.K. Rayala, R. Kumar, MicroRNA-7, a homeobox D10 target, inhibits p21-activated kinase 1 and regulates its functions, Cancer Res, 68 (2008) 8195-8200. [58] O. Saydam, O. Senol, T. Wurdinger, A. Mizrak, G.B. Ozdener, A.O. Stemmer-Rachamimov, M. Yi, R.M. Stephens, A.M. Krichevsky, N. Saydam, G.J. Brenner, X.O. Breakefield, miRNA-7 attenuation in Schwannoma tumors stimulates growth by upregulating three oncogenic signaling pathways, Cancer Res, 71 (2011) 852-861. [59] R.J. Webster, K.M. Giles, K.J. Price, P.M. Zhang, J.S. Mattick, P.J. Leedman, Regulation of epidermal growth factor receptor signaling in human cancer cells by microRNA-7, J Biol Chem, 284 (2009) 5731-5741. [60] S. Xiong, Y. Zheng, P. Jiang, R. Liu, X. Liu, Y. Chu, MicroRNA-7 inhibits the growth of human non-small cell lung cancer A549 cells through targeting BCL-2, Int J Biol Sci, 7 (2011) 805-814. [61] H. Okuda, F. Xing, P.R. Pandey, S. Sharma, M. Watabe, S.K. Pai, Y.Y. Mo, M. Iiizumi-Gairani, S. Hirota, Y. Liu, K. Wu, R. Pochampally, K. Watabe, miR-7 suppresses brain metastasis of breast cancer stem-like cells by modulating KLF4, Cancer Res, 73 (2013) 1434-1444.
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[62] L. Jiang, X. Liu, Z. Chen, Y. Jin, C.E. Heidbreder, A. Kolokythas, A. Wang, Y. Dai, X. Zhou, MicroRNA-7 targets IGF1R (insulin-like growth factor 1 receptor) in tongue squamous cell carcinoma cells, Biochem J, 432 (2010) 199-205. [63] X. Zhao, W. Dou, L. He, S. Liang, J. Tie, C. Liu, T. Li, Y. Lu, P. Mo, Y. Shi, K. Wu, Y. Nie, D. Fan, MicroRNA-7 functions as an anti-metastatic microRNA in gastric cancer by targeting insulin-like growth factor-1 receptor, Oncogene, 32 (2013) 1363-1372. [64] X. Kong, G. Li, Y. Yuan, Y. He, X. Wu, W. Zhang, Z. Wu, T. Chen, W. Wu, P.E. Lobie, T. Zhu, MicroRNA-7 inhibits epithelial-to-mesenchymal transition and metastasis of breast cancer cells via targeting FAK expression, PLoS One, 7 (2012) e41523. [65] B. Wang, F. Sun, N. Dong, Z. Sun, Y. Diao, C. Zheng, J. Sun, Y. Yang, D. Jiang, MicroRNA-7 directly targets insulin-like growth factor 1 receptor to inhibit cellular growth and glucose metabolism in gliomas, Diagn Pathol, 9 (2014) 211. [66] N. Zhang, X. Li, C.W. Wu, Y. Dong, M. Cai, M.T. Mok, H. Wang, J. Chen, S.S. Ng, M. Chen, J.J. Sung, J. Yu, microRNA-7 is a novel inhibitor of YY1 contributing to colorectal tumorigenesis, Oncogene, 32 (2013) 5078-5088. [67] Y.T. Chou, H.H. Lin, Y.C. Lien, Y.H. Wang, C.F. Hong, Y.R. Kao, S.C. Lin, Y.C. Chang, S.Y. Lin, S.J. Chen, H.C. Chen, S.D. Yeh, C.W. Wu, EGFR promotes lung tumorigenesis by activating miR-7 through a Ras/ERK/Myc pathway that targets the Ets2 transcriptional repressor ERF, Cancer Res, 70 (2010) 8822-8831. [68] A.M. Cheng, M.W. Byrom, J. Shelton, L.P. Ford, Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis, Nucleic Acids Res, 33 (2005) 1290-1297. [69] Z. Yu, L. Ni, D. Chen, Q. Zhang, Z. Su, Y. Wang, W. Yu, X. Wu, J. Ye, S. Yang, Y. Lai, X. Li, Identification of miR-7 as an oncogene in renal cell carcinoma, J Mol Histol, 44 (2013) 669-677. [70] M.S. Ebert, J.R. Neilson, P.A. Sharp, MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells, Nat Methods, 4 (2007) 721-726. [71] M.S. Ebert, P.A. Sharp, MicroRNA sponges: progress and possibilities, RNA, 16 (2010) 2043-2050. [72] C.W. Lin, Y.L. Chang, Y.C. Chang, J.C. Lin, C.C. Chen, S.H. Pan, C.T. Wu, H.Y. Chen, S.C. Yang, T.M. Hong, P.C. Yang, MicroRNA-135b promotes lung cancer metastasis by regulating multiple targets in the Hippo pathway and LZTS1, Nat Commun, 4 (2013) 1877. [73] Y. Liu, Y. Han, H. Zhang, L. Nie, Z. Jiang, P. Fa, Y. Gui, Z. Cai, Synthetic miRNA-mowers targeting miR-183-96-182 cluster or miR-210 inhibit growth and migration and induce apoptosis in bladder cancer cells, PLoS One, 7 (2012) e52280.
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Figure 1. Biogenesis of circRNAs.
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Exon-skipping of pre-mRNA produces linear mRNAs and additional remnants form
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circles, or exonic circRNAs. Introns with complementary ALU repeats form circles
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through base-pairing to make ciRNA (circular intron RNAs). CircRNAs that contain
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both exons and introns are RIciRNA (Exon-Intron circRNA).
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Figure 2. Functions of circRNAs.
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(A) Acting as an miRNA sponge or ceRNA. (B) Directly targeting mRNA by partly
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base pairing. (C) Binding RNA binding protein (RBP) and AGO to regulate protein
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expression. (D) Used as the template of protein synthesis. IRES: Internal ribosome
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entry site.
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Figure 3. CDR1as /miR-7 and tumor.
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(A) CDR1as functions as an miR-7 buffer to inhibit the expression of miR-7’s targets.
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(B) The balance of CDR1as /miR-7 impacts a number of signaling pathways to
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regulate tumorigenesis, development and metastasis. EGFR: epidermal growth factor
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receptor; IRS-1/2: insulin receptor substrate-1/2; Pak1: p21-activated kinase-1; Ack1:
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activated CDC42 kinase 1; PIK3CD: phosphatidylinositol 3-kinase catalytic subunit δ;
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mTOR: mammalian target of rapamycin; IGF1R: insulin-like growth factor 1 receptor;
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KLF4: Kruppel-like factor 4; EMT: epithelial-to-mesenchymal transition; CSC:
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Highlights: 1. circRNA is exonic, intronic or retained-intronic. 2. CircRNAs mediate transcriptional and post-transcriptional regulation of gene and protein expression. 3. CircRNAs are critical for cancer biology.
S0304-3835(17)30201-X
DOI:
10.1016/j.canlet.2017.03.027
Reference:
CAN 13291
To appear in:
Cancer Letters
Received Date: 15 February 2017 Revised Date:
15 March 2017
Accepted Date: 15 March 2017
Please cite this article as: J. He, Q. Xie, H. Xu, J. Li, Y. Li, Circular RNAs and Cancer, Cancer Letters (2017), doi: 10.1016/j.canlet.2017.03.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Circular RNAs and Cancer
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Jun He1,#,*, Qichao Xie2,#, Hailin Xu1, Jiantian Li1, Yongsheng Li2,*
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1 Department of General Surgery, Jiande Branch of The Second Affiliated Hospital,
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School of Medicine, Zhejiang University, Jiande, Zhejiang Province 311600, China.
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2 Institute of Cancer, Xinqiao Hospital, Third Military Medical University,
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Chongqing 400037, China.
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#J.H. and Q.X. contributed equally to this manuscript and share the first authorship. *Correspondence: [email protected] (J. He); [email protected] (Y. Li).
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Word count of the text: 3519.
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Abstract
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Circular RNAs (circRNAs) are a type of non-coding RNA molecules that lack a
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5’-terminal cap and 3’-terminal poly A tail. A large number of circRNAs have been
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identified
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high-throughput sequencing. CircRNA sequence composition determines if a given
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circRNA is exonic, intronic or retained-intronic. CircRNAs are more abundant and
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stable than linear mRNAs, and their expression is both step- and location-specific.
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CircRNAs mediate transcriptional and post-transcriptional regulation of gene and
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protein expression. CircRNAs regulate cancer development via multiple mechanisms,
biological
experiments,
computational
methods
and
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including
sponges,
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epithelial-mesenchymal transition. An in-depth study of circRNA will provide a better
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understanding of carcinogenesis and assist in developing clinical diagnostic and
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therapeutic strategies.
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regulation
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Keywords: circRNA; cancer; miRNA sponge; transcription.
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signaling
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the
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1 Introduction Circular RNAs (circRNAs) are a new class of long non-coding RNA which do
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not have 5’ or 3’ ends but are covalently linked to form a closed circular structure.
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Their presence was firstly observed by Sanger in a virus using electron microscopy
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more than 40 years ago [1]. Later, circRNAs in humans, mice, fungi and other
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organisms were subsequently found [2-4]. Due to the structure specificity and low
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abundance, circRNAs were only identified in a few individual genes, including ETS-1
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[5], Sex-determining region Y (SRY) [6], cytochrome P450 2C24 and 2C18 [7, 8] and
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cANRIL (circular ANRIL) [9] over subsequent decades. They were considered as an
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ancient and conserved class of molecules and are the abnormal splicing products of
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RNAs, or the “dark matter” in organisms [10].
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In recent years, molecular purification methods combined with high-throughput
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sequencing technology and improvement of statistical calculation [11] have led to an
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in-depth understanding of these “dark matter”. Specific algorithms and methods for
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use in circRNA research include RNA-seq [12, 13], ssRNA-seq [14], MapSplice [15],
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CircSeq [12], circBase [16], PFOR2 [17] CIRI [18] and segemehl [19]. Using the
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above methods, more than 20,000 circRNAs have been predicted, but they need to be
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verified [12, 20]. Recently, Arraystar pioneered the development of the world’s first
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commercial circRNA chip, which provides an experimental platform for
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systematically characterizing the expression of circRNA in different physiological and
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pathological conditions [21]. Consequently, the development of circRNA in research
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lays a foundation of further exploration of their generation and biofunction in cancer.
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2 Mechanisms of circRNA maturation CircRNAs can be divided into the following three categories based on their
genomic origin and sequence composition (Figure 1).
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2.1 Exonic circRNAs
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Exonic circRNAs are the most abundant circRNAs. Most exonic circRNAs are
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generated from coding genes but do not encode proteins [12, 22, 23]. Generally, 3
ACCEPTED MANUSCRIPT non-coding introns in eukaryotic genes are removed by alternative splicing of
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pre-mRNAs after transcription, followed by sequential linking of exons containing
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protein-coding information to form mature linear RNAs and corresponding protein
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products, this process is called sequential splicing. However, the exonic circRNAs are
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formed by back-splicing, i.e. exon sequences of genes are linked reverse end-to-end.
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There are currently two potential models of back-splicing [12]: lariat-driven
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circularization and intron-pairing-driven circularization. The former suggests that
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during transcription of pre-RNA, partial splicing of the RNA (i.e., covalent binding of
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the splice donor of one exon to the splice acceptor of a different exon narrows the
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distances between the original non-adjacent exons) leads to exon skipping and the
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formation of a circular RNA intermediate. Subsequently, exonic circRNA is formed
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by intra-lariat splicing.
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The intron-pairing-driven circularization model has been extensively validated.
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This model indicates that exons involved in circularization are connected to introns
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containing reverse complementation sequences that result in spatially close donor and
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acceptor splice exons that form circRNAs. In the 1990s, it was discovered that
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circularization of circEts-1 and circSry driven by complementary sequence pairing [5].
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It should be noted, however, that not all circRNAs are generated by complementary
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regions on either side of an exon. For example, Wang et al. constructed a minigene in
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vitro and found that the reverse complementation of exon-flanking introns upstream
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and downstream is not necessary for the formation of circRNAs [24]. In 2014,
22
Ashwal-Fluss et al. identified an antagonistic relationship between linear mRNA
23
splicing and back splicing of circRNAs [25]. Pre-mRNAs containing stable 3’ ends
24
were more likely to form circRNAs [26], suggesting that circRNAs could be produced
25
after transcription rather than during co-transcription, as has been described for
26
eukaryotic pre-mRNAs.
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One common feature was noted in both of the models described, i.e., long
28
flanking introns of complementary ALU repeats [12]. The exon circularization is
29
mediated by intron-pairing introns [27]. Human genome intron regions contain a large
30
number of complementary sequences whose pairing can also produce multiple 4
ACCEPTED MANUSCRIPT 1
circRNAs through a mechanism called alternative circularization.
2 3
2.2 CiRNAs Introns account for more than 20% of the human genome, where most introns
5
form a lasso structure after splicing that is degraded after off-branch [28]. However,
6
some introns containing certain key nucleic acid sequences cannot debranch after
7
splicing and instead form intron-derived ciRNAs. It is known that the production of
8
ciRNAs is dependent on conserved motifs at both ends of the intron, i.e., the
9
7-nucleotide GU-rich motif located at the 5’ splicing site and the 11-nucleotide C-rich
10
motif at 3’-branch site. These nucleotides prevent debranching and cause the RNA to
11
retain a circular structure [27]. Potential additional protein factors involved in the
12
formation of ciRNAs are unknown. The distinct method of formation of ciRNAs
13
results in distinct differences from exonic circRNAs: ciRNAs are 2’-5’
14
phospholipid-linked molecules, and exonic circRNAs are 3’-5’ phospholipid-linked
15
molecules. CiRNAs are present in the nucleus and are able to bind to and regulate the
16
expression of parent genes.
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2.3 Retained-intron circRNAs
In the process of back-splicing to form exonic circRNAs, circular RNAs
20
containing un-spliced introns can stabilize and exist [20], potentially acting as an
21
intermediate in the splicing process, or to form distinct circRNAs. By preparing RNA
22
polymerase (Pol)
23
immunoprecipitating with Pol II antibody) followed by RNA sequencing and
24
bioinformatics analysis, Li et al. found more than 100 circRNAs interacting with Pol
25
II in human cells [29]. Further studies have shown that these circular RNAs are also
26
formed by exon back-splicing, but this type of circRNAs contain both exons and
27
introns and are fully localized to the nucleus. Hence, these specific circRNAs were
28
termed exon-intron circRNAs (EIciRNAs).
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3 CircRNA biofunctions 5
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RNA and
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3.1 As a ceRNA or miRNA sponge Competitive endogenous RNA (ceRNA) contains a miRNA response element
3
(MRE), which competitively binds miRNA [30, 31]. Therefore, ceRNA can affect the
4
regulatory functions of miRNAs in gene expression and reduce the inhibitory effect of
5
miRNAs on target molecules. Recent studies have shown that exonic circRNAs can
6
act as ceRNA or miRNA sponge molecules, which regulate the expression of genes by
7
adsorbing miRNAs (Figure 2A) [32]. CDR1as (antisense to the cerebellar
8
degeneration-related protein 1 transcript) is a natural antisense transcript (NAT) of the
9
cerebellar degeneration-associated protein 1 (CDR1) gene. This circRNA is
10
approximately 1.5 kb in length and contains 74 binding sites for miR-7. More than 60
11
sites are conserved and are capable of binding to RNA-induced silencing complexes
12
(RISC) formed by miR-7 and Ago2 protein [33]. Therefore, CDR1as is also called
13
ciRS-7 (circular RNA sponge for miR-7). The circRNA CDR1as co-localizes with
14
miR-7 in the cytoplasm. Knockdown of CDR1as or overexpression of miR-7
15
promotes the degradation of miR-7-target mRNAs. Conversely, overexpression of
16
CDR1as inhibits the action of miR-7 and can mimic the phenotype of morpholino
17
miR-7 knockdown, leading to a decrease of the midbrain size in embryos of zebrafish
18
[13]. Hansen et al. found that although CDR1as cannot be degraded by
19
miR-7-mediated RISC, it can complement miR-671 and degrade [34], suggesting that
20
miR-671 indirectly regulates miR-7 by lowering CDR1as.
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SRY gene consists of only one exon. In the early stages of development, its
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transcript forms a linear RNA, serving as a template for protein synthesis. However,
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in adult testes, its cytoplasmic RNA is mainly circular and does not undergo
24
translation [6]. Studies have confirmed that the reverse repeat sequence on both sides
25
of the SRY exon can be directly transcribed into a circRNA molecule [35, 36]. The
26
circular transcript of SRY gene has a similar function to CDR1as, in that it contains 16
27
MREs of miRNA-138 and displays as the miR-138 sponge, thereby regulating the
28
expression of miR-138-target genes [37]. In addition, Li et al. found that circ-ITCH
29
could be used as a sponge of miR-7, miR-17 and miR-214 [38]. Bioinformatics
30
analysis has shown that tens of thousands of circular RNAs have miRNA adsorption
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functional capabilities [39], but few have been validated. Therefore, universal
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circRNA sponge function and regulation of miRNAs and ceRNAs requires
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elucidation.
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3.2 Regulating gene transcription
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Recent studies have shown that circRNA can regulate parental gene expression.
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For example, The formation of circRNAs is dependent on key flanking RNA elements
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that are critical for intron lasso-circularization eluting debranching [27]. These
9
circRNAs do not enrich miRNA targets, indicating that their functions are unique.
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CircRNAs that regulate the transcription of parental genes include some ciRNAs (e.g.,
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ci-ankrd52, ci-sirt7) and EIciRNAs (e.g., circEIF3J, circPAIP2).
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Detailed DNA/RNA duplex in situ hybridization showed that some ciRNAs are
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localized to their transcriptional sites and can bind RNA pol II complexes to affect the
14
transcription of RNA pol II via an unknown mechanism, thereby cis-regulating their
15
parent genes (Figure 2B). Interestingly, ciRNAs not only are enriched at their
16
transcriptional sites but also accumulate in other regions of the nucleus, suggesting
17
that they may play a trans-regulatory role. Ci-ankrd52 is an ankrd52-derived ciRNA
18
that specifically binds RNA pol II actively transcribed by its parent gene ankrd52 to
19
regulate the transcription efficiency of ankrd52. Upon ci-ankrd52 knock out, ankrd52
20
transcription efficiency is significantly reduced, however, administration of high
21
levels of ci-ankrd did not improve the transcription efficiency of ankrd52.
22
Researchers speculate that this may be caused by the abnormal positioning of
23
exogenous ci-ankrd52 [27]. Silent information regulator 7 (ci-sirt7) also acts via a
24
similar mechanism [27]. In addition, cANRIL, an exonic circRNA, is also presumed
25
have transcriptional regulation activity. ANRIL inhibits transcription of its encoding
26
gene INK4/ARF by binding to the Polycomb Gene (PcG) complex. ANRIL transcripts
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can undergo back-splicing to form cANRIL, suggesting that formation of cANRIL
28
reduces ANRIL, thus regulating transcription of INK4/ARF [9].
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Studies of two EIciRNAs, namely, EIciEIF3J and EIciPAIP2, show that both
30
co-localize with their parental locus and U1 snRNP (small nuclear ribonucleoprotein), 7
ACCEPTED MANUSCRIPT bind to U1 snRNA (small nuclear RNA) and recruit U1 snRNP, promoting the
2
interaction of RNA pol II with the promoter region of the parental gene resulting in in
3
cis transcription initiation of the parent gene. Consistently, 3’ untranslated regions
4
(3’-UTR) of cir-ITCH and ITCH have been found to share miRNA binding sites [29].
5
Further studies have demonstrated that cir-ITCH interacts with miR-7, miR-17 and
6
miR-214 to up-regulate ITCH expression [38]. It is speculated that exon-derived
7
circRNAs play a regulatory role in the cytoplasm, while intron-derived circRNAs
8
(such as ciRNA and EIciRNA) play a transcriptional regulatory role in the nucleus.
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3.3 Regulating RNA binding proteins
The adsorption of protein factors by linear long non-coding RNA (lncRNA) has
12
been reported. For example, sno-lncRNAs regulate alternative splicing of downstream
13
genes by adsorbing the alternative splicing factor Fox2 [40]. CircRNA molecules can
14
also adsorb protein factors. RNA splicing factor MBL can bind exon 2 of its parental
15
gene and promote its circularization to form circMBL. CircMBL can then bind MBL,
16
reducing the effective concentration of MBL and the production of circMBL [25]. The
17
binding of intron-exon circular RNA circEIF3J with U1 RNA promotes the binding of
18
U1 snRNP complex and RNA pol II and enhances parental gene transcription (Figure
19
2C) [29]. The complex formation of circ-Foxo3, kinase inhibitor protein (p21) and
20
cell division protein kinase 2 (CDK2) inhibits the promotion of CDK2 on cell division
21
and blunts cell cycle progression [41]. CDR1as and circSry can bind with the miRNA
22
effector AGO to cleave it, inhibit its translation, and eventually promotes its
23
degradation (Figure 2C) [13, 33]. Ci-ankrd52 can interact with the RNA pol II
24
complex to affect its activity and ultimately regulate transcription [27]. These findings
25
suggest that circRNAs can serve as a scaffolding platform for protein-RNA,
26
protein-DNA, and protein-protein interactions.
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3.4 Protein translation
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CircRNAs can also be translated to proteins, similar to linear mRNAs. In 1995,
30
Chen and Sarnow et al. found that an internal ribosome entry site (IRES) and the 8
ACCEPTED MANUSCRIPT initiation codon ATG in a specific circRNA would allow the circRNA translation
2
template to function as mRNA (Figure 2D) [42]. In 2014, Jeck et al. found that many
3
exon-derived circRNAs contain translation initiation sites. CircRNAs regulate protein
4
expression by blocking the translation initiation site [43]. Wang et al. constructed a
5
minigene in vitro containing the cytomegalovirus (CMV) promoter, IRES, and an
6
exon encoding green fluorescent protein (GFP), which allowed the circularization of
7
corresponding transcript [24]. After transfection into cells, this minigene transcript
8
was able to form circular RNA that was capable of translating GFP protein.
9
Interestingly, to date, only one naturally occurring circRNA can encode proteins in
10
eukaryotic cells, namely hepatitis D virus (HDV). HDV is a satellite virus of HBV
11
(hepatitis B virus). HDV and HBV virus particles co-embedded and produce the same
12
protein; however, translation of the protein is not regular, likely due to the viral vector.
13
The role of circRNAs in control of translation still warrants further study [44].
14 15
4 CircRNAs in cancer
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Studies have shown a close relationship between circRNAs and a variety of
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tumors, including colon cancer, ovarian cancer, gastric cancer, esophageal cancer, and
18
glioma [38, 45-49]. The wide existence, high stability and variety of regulatory
19
functions of circRNAs are undoubtedly a novel area of interest in the early diagnosis
20
and treatment of cancer. Some clinical studies have shown that the levels of certain
21
circRNAs are decreased in tumor tissue in comparison to normal tissue and that
22
circRNA levels are significantly correlated with clinical features of distant metastasis,
23
staging, age of onset, and gender. CircRNA expression in tumor tissue and tumor cell
24
lines were found reduced, and the ratio of specific circRNAs and their linear isomers
25
was significantly different in healthy tissue and that affected by colorectal cancer
26
when analyzed by transcriptome analysis [50], providing a new molecular focus in the
27
study of pathogenesis of tumors. Many tumor-associated chromosomal translocation
28
regions produce fusion circRNAs (f-circRNAs) , which might promote cancer growth
29
[51]. CircRNAs are also enriched in exosomes [52], indicating that it might be
30
possible to diagnose cancer by detecting serum circRNAs.
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4.1 Cancer biology-related circRNA signal pathway CircRNAs are involved in the regulation of the molecular biology of tumors via
4
multiple mechanisms. For instance The expression of circ-ITCH in human esophageal
5
cancer tissues is lower than that in para-cancerous tissue. Circ-ITCH can inhibit the
6
activity of the Wnt pathway in the esophageal cancer cell lines Eca-109 and TE-1,
7
resulting in inhibition of the proliferative ability, cell cycle progression and
8
tumorigenic ability [38]. In addition, circRNAs also regulate epithelial-mesenchymal
9
transition (EMT). During the process of EMT, QUAKING protein can bind to the
10
flanking sequence of specific circRNAs and regulate the production of these
11
circRNAs. Of note, the number of such circRNA has reached thousands, suggesting
12
an inextricable link between circRNAs and cancer development [53].
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4.2. CircRNA and cancer-related miRNA
Hansen originally proposed the idea that circRNAs can target miRNAs [32].
16
Recently, circRNAs were shown to play an important role in the regulation of
17
miRNA-mediated gene expression by isolation of miRNAs. Subsequently, Ghosal et
18
al. performed gene cluster analysis (GO) and analyzed protein-coding loci of
19
miRNA-circRNA-related diseases to detect gene enrichment associated with specific
20
physiological processes [54]. This was the first comprehensive data analysis of the
21
role of circRNA in cancer, however the relationship between circRNA-miRNAs
22
requires investigation.
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CDR1as/miR-7 can affect carcinogenesis and progression of disease in a variety
24
of ways (Figure 3A) [34]. Human ciRS-7 is highly expressed in cells and interacts
25
with
26
miRNA-mediated degradation and significantly inhibit miR-7 activity, thereby
27
up-regulating miR-7 target gene expression [32]. Owing to the high expression of
28
ciRS-7 and miR-7 in brain tissue, the frequent ciRS-7/miR-7 interaction will change
29
intracellular levels of RISC. Therefore, miRNAs and miRNA regulatory activity in
30
miR-7/ciRS-7 overexpressing tissues are inactive [34].
AGO
protein
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a
miR-7
dependent
10
manner.
CircRNAs
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ACCEPTED MANUSCRIPT Some miR-7 targets are important proteins in cancer-related signaling pathways,
2
such as epidermal growth factor receptor (EGFR), insulin receptor substrate-1 (IRS-1),
3
activated CDC42 kinase 1 (Ack1), and phosphatidylinositol 3-kinase catalytic subunit
4
δ (PIK3CD) (Figure 3B) [55-59]. MiR-7 can effectively inhibit the expression of
5
EGFR and reduce the expression of IRS-1 and IRS-2 by inhibiting protein kinase B
6
(PKB) resulting in reduced activity and invasion of glioma cells [56]. Pak1 is a
7
member of serine threonine protein kinases. Endogenous miR-7 is negatively
8
correlated with levels of Pak1 and positively correlated with Homeobox D10
9
(HOXD10), a homology domain transcription factor. During the transformation of
10
low-invasive to highly invasive breast cancer, expression of Pak1 was up-regulated
11
while the expression of miR-7 and HOXD10 were down-regulated. In highly
12
aggressive breast cancer cells, miR-7 inhibited the proliferative activity, invasion and
13
tumorigenic potential. These results indicate that miR-7/Pak1 pathway plays an
14
important role in the development of breast cancer [57]. In lung cancer, breast cancer
15
and glioblastoma cell lines, miR-7 significantly reduces the expression of
16
EGFR-related mRNAs, including Raf1, protein kinase B/Akt (PKB/Akt) and
17
extracellular signal-regulated kinase (ERK) 1/2 [59]. In schwannoma cells, Ack1 is a
18
direct target of miR-7, and its expression is inversely proportional to that of miR-7
19
[58]. In vitro, miR-7 overexpression is involved in cell cycle arrest and cell migration
20
regulated by the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway.
21
Additionally the expression of Akt, mammalian target of rapamycin (mTOR) and
22
p70S6K are down-regulated in HCC cells [55]. MiR-7 inhibits the growth of human
23
non-small cell lung cancer A549 cells by regulating the expression of B-cell
24
lymphoma-2 (BCL-2), an apoptosis-related gene. Cancer stem cells (CSC) play an
25
important role in cancer progression and metastasis. It has been confirmed that miR-7
26
is downregulated compared to all other miRNAs in CSC [60]. Kruppel-like factor 4
27
(KLF4) is a key transcription factor in CSC. The anticancer effect of miR-7 may be
28
partly due to its concomitant inhibition of KLF4, which plays an important regulatory
29
role in metastasis of breast cancer [61]. In addition, miR-7 up-regulates the expression
30
of cadherin to indirectly inhibit EMT by suppressing insulin-like growth factor 1
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receptor (IGF1R) and focal adhesion kinase, resulting in delayed tumor growth and
2
metastasis. This mechanism is important in the tumorigenesis of gastric cancer,
3
squamous cell carcinoma, breast cancer and malignant glioma [62-65]. However, some studies reported a cancer-promoting role of miR-7. For example,
5
miR-7 down-regulated compared to all other miRNAs in colorectal cancer. It targets
6
oncogene YY1, leading to P53 inactivation [66]. However, the expression of CDR1as
7
is significantly increased in colorectal cancer tissues. In the lung cancer CL1-5 cell
8
line, miR-7 levels are positively correlated with increased transplanted tumor volume
9
and the survival rate of nude mice [67]. In cervical cancer and lung adenocarcinoma
10
cell lines, inhibition of miR-7 resulted in decreased proliferation and increased
11
apoptosis, suggesting that overexpression of miR-7 may not lead to suppression of
12
carcinogenesis [68]. This is also shown in renal cell carcinoma, where invasion and
13
proliferation are associated with high expression of miR-7 [69]. Altogether, miR-7 is
14
closely related to ciRS-7, and the micro-regulatory axis of miR-7/miR-671/ciRS-7
15
may play an important role in cancer pathophysiology.
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4.3 CircRNA and cancer therapy
Use of gene knockout, antisense oligonucleotides and miRNA sponges are three
19
classical approaches to cause loss of miRNA function [70]. Construction of knockout
20
animal models is time-consuming, expensive and difficult. Chemically modified
21
antisense oligonucleotides are useful in short-term experiments, but cannot achieve
22
long-term miRNA inhibition. miRNA sponge technology is a new technology and
23
possible alternative to gene knockout technology [71]. miRNA sponges transfected
24
into human cells have similar miRNA inhibiting potency to antisense oligonucleotides.
25
Compared with conventional linear miRNA sponges containing a single MRE,
26
circRNA sponges contain several MREs. In malignant melanoma cell lines, circRNA
27
sponges exhibited superior anticancer effects when compared to linear sponges.
28
Circular sponges may be the best intracellular means of inhibiting miRNA activity.
29
Importantly, these sponges could be used to target oncogenes and reverse the
30
malignant phenotype of human cancer cells [72]. Artificial sponges are a powerful
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ACCEPTED MANUSCRIPT treatment option to target RNA. For example, using T4 phage gene td self-splicing,
2
Liu et al. constructed a circRNA sponge targeting miR-21 and miR-221 and used it to
3
treat melanoma cell lines. The results demonstrate that the circRNA sponge showed
4
more potent anti-cancer effect than linear RNA [73]. These findings suggest that
5
synthetic circRNA inhibitors are a novel approach for future cancer therapies.
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5 Concluding remarks
With improvements in the new generation of high-throughput sequencing and
9
biological technologies, different researchers continue to gain an in-depth
10
understanding of circRNAs. These molecules act as an miRNA molecule sponge,
11
interact with RNA-binding proteins, and participate in gene transcriptional regulation.
12
However, there are many other unknown functional mechanisms to be investigated.
13
The identification and functional study of these new RNA molecules not only enrich
14
our understanding of the complexity of the eukaryotic transcriptome and non-coding
15
RNA but also provide new ideas and methods for the diagnosis and treatment of
16
human diseases. We can appropriately modify circular RNA molecules to silence
17
important binding sites associated with cancer. We can also target specific molecular
18
drugs to change downstream gene expression in order to treat cancer. At the same
19
time, emerging and improved methods of artificially constructing circular RNAs or
20
interfering RNAs makes it possible to regulate the expression of intracellular
21
circRNAs, which is essential to further explore the function of circRNAs [13, 53].
22
The use of circRNAs in clinical diagnosis and treatment has become a new foothold
23
for translational and precision medicine. The appropriate and precise use referring to
24
circRNAs and understanding their functionalities and mechanisms are the key topics
25
in the field of circRNAs and cancer research in the years to come. The continuous
26
exploration and research in this field will provide an important molecular basis for
27
understanding the complex regulation of life activities.
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ACCEPTED MANUSCRIPT Conflicts of Interest: The authors have declared no conflicts of interest
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Acknowledgements: The work was supported by Youth 1000 Talent Plan and the
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National Natural Science Foundation of China (81472435 and 81671573).
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[1] H.L. Sanger, G. Klotz, D. Riesner, H.J. Gross, A.K. Kleinschmidt, Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures, Proc Natl Acad Sci U S A, 73 (1976) 3852-3856. [2] D. Kolakofsky, Isolation and characterization of Sendai virus DI-RNAs, Cell, 8 (1976) 547-555. [3] Y. Matsumoto, R. Fishel, R.B. Wickner, Circular single-stranded RNA replicon in Saccharomyces cerevisiae, Proc Natl Acad Sci U S A, 87 (1990) 7628-7632. [4] J.M. Nigro, K.R. Cho, E.R. Fearon, S.E. Kern, J.M. Ruppert, J.D. Oliner, K.W. Kinzler, B. Vogelstein, Scrambled exons, Cell, 64 (1991) 607-613. [5] C. Cocquerelle, P. Daubersies, M.A. Majerus, J.P. Kerckaert, B. Bailleul, Splicing with inverted order of exons occurs proximal to large introns, EMBO J, 11 (1992) 1095-1098. [6] B. Capel, A. Swain, S. Nicolis, A. Hacker, M. Walter, P. Koopman, P. Goodfellow, R. Lovell-Badge, Circular transcripts of the testis-determining gene Sry in adult mouse testis, Cell, 73 (1993) 1019-1030. [7] P.G. Zaphiropoulos, Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping, Proc Natl Acad Sci U S A, 93 (1996) 6536-6541. [8] P.G. Zaphiropoulos, Exon skipping and circular RNA formation in transcripts of the human cytochrome P-450 2C18 gene in epidermis and of the rat androgen binding protein gene in testis, Mol Cell Biol, 17 (1997) 2985-2993. [9] C.E. Burd, W.R. Jeck, Y. Liu, H.K. Sanoff, Z. Wang, N.E. Sharpless, Expression of linear and novel circular forms of an INK4/ARF-associated non-coding RNA correlates with atherosclerosis risk, PLoS Genet, 6 (2010) e1001233. [10] C. Cocquerelle, B. Mascrez, D. Hetuin, B. Bailleul, Mis-splicing yields circular RNA molecules, FASEB J, 7 (1993) 155-160. [11] F. Caiment, S. Gaj, S. Claessen, J. Kleinjans, High-throughput data integration of RNA-miRNA-circRNA reveals novel insights into mechanisms of benzo[a]pyrene-induced carcinogenicity, Nucleic Acids Res, 43 (2015) 2525-2534. [12] W.R. Jeck, J.A. Sorrentino, K. Wang, M.K. Slevin, C.E. Burd, J. Liu, W.F. Marzluff, N.E. Sharpless, Circular RNAs are abundant, conserved, and associated with ALU repeats, RNA, 19 (2013) 141-157. [13] S. Memczak, M. Jens, A. Elefsinioti, F. Torti, J. Krueger, A. Rybak, L. Maier, S.D. Mackowiak, L.H. Gregersen, M. Munschauer, A. Loewer, U. Ziebold, M. Landthaler, C. Kocks, F. le Noble, N. Rajewsky, Circular RNAs are a large class of animal RNAs with regulatory potency, Nature, 495 (2013) 333-338. [14] T. Lu, L. Cui, Y. Zhou, C. Zhu, D. Fan, H. Gong, Q. Zhao, C. Zhou, Y. Zhao, D. Lu, J. Luo, Y. Wang, Q. Tian, Q. Feng, T. Huang, B. Han, Transcriptome-wide investigation of circular RNAs in rice, RNA, 21 (2015) 2076-2087. [15] K. Wang, D. Singh, Z. Zeng, S.J. Coleman, Y. Huang, G.L. Savich, X. He, P. Mieczkowski, S.A. Grimm, C.M. Perou, J.N. MacLeod, D.Y. Chiang, J.F. Prins, J. Liu, MapSplice: accurate mapping of RNA-seq reads for splice junction discovery, Nucleic Acids Res, 38 (2010) e178.
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References
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[16] P. Glazar, P. Papavasileiou, N. Rajewsky, circBase: a database for circular RNAs, RNA, 20 (2014) 1666-1670. [17] Z. Zhang, S. Qi, N. Tang, X. Zhang, S. Chen, P. Zhu, L. Ma, J. Cheng, Y. Xu, M. Lu, H. Wang, S.W. Ding, S. Li, Q. Wu, Discovery of replicating circular RNAs by RNA-seq and computational algorithms, PLoS Pathog, 10 (2014) e1004553. [18] Y. Gao, J. Wang, F. Zhao, CIRI: an efficient and unbiased algorithm for de novo circular RNA identification, Genome Biol, 16 (2015) 4. [19] S. Hoffmann, C. Otto, G. Doose, A. Tanzer, D. Langenberger, S. Christ, M. Kunz, L.M. Holdt, D. Teupser, J. Hackermuller, P.F. Stadler, A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection, Genome Biol, 15 (2014) R34. [20] J. Salzman, R.E. Chen, M.N. Olsen, P.L. Wang, P.O. Brown, Cell-type specific features of circular RNA expression, PLoS Genet, 9 (2013) e1003777. [21] X. Fan, X. Zhang, X. Wu, H. Guo, Y. Hu, F. Tang, Y. Huang, Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos, Genome Biol, 16 (2015) 148. [22] J. Salzman, C. Gawad, P.L. Wang, N. Lacayo, P.O. Brown, Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types, PLoS One, 7 (2012) e30733. [23] J.U. Guo, V. Agarwal, H. Guo, D.P. Bartel, Expanded identification and characterization of mammalian circular RNAs, Genome Biol, 15 (2014) 409. [24] Y. Wang, Z. Wang, Efficient backsplicing produces translatable circular mRNAs, RNA, 21 (2015) 172-179. [25] R. Ashwal-Fluss, M. Meyer, N.R. Pamudurti, A. Ivanov, O. Bartok, M. Hanan, N. Evantal, S. Memczak, N. Rajewsky, S. Kadener, circRNA biogenesis competes with pre-mRNA splicing, Mol Cell, 56 (2014) 55-66. [26] D. Liang, J.E. Wilusz, Short intronic repeat sequences facilitate circular RNA production, Genes Dev, 28 (2014) 2233-2247. [27] Y. Zhang, X.O. Zhang, T. Chen, J.F. Xiang, Q.F. Yin, Y.H. Xing, S. Zhu, L. Yang, L.L. Chen, Circular intronic long noncoding RNAs, Mol Cell, 51 (2013) 792-806. [28] F. Rodriguez-Trelles, R. Tarrio, F.J. Ayala, Origins and evolution of spliceosomal introns, Annu Rev Genet, 40 (2006) 47-76. [29] Z. Li, C. Huang, C. Bao, L. Chen, M. Lin, X. Wang, G. Zhong, B. Yu, W. Hu, L. Dai, P. Zhu, Z. Chang, Q. Wu, Y. Zhao, Y. Jia, P. Xu, H. Liu, G. Shan, Exon-intron circular RNAs regulate transcription in the nucleus, Nat Struct Mol Biol, 22 (2015) 256-264. [30] L. Poliseno, L. Salmena, J. Zhang, B. Carver, W.J. Haveman, P.P. Pandolfi, A coding-independent function of gene and pseudogene mRNAs regulates tumour biology, Nature, 465 (2010) 1033-1038. [31] M. Cesana, D. Cacchiarelli, I. Legnini, T. Santini, O. Sthandier, M. Chinappi, A. Tramontano, I. Bozzoni, A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA, Cell, 147 (2011) 358-369. [32] T.B. Hansen, T.I. Jensen, B.H. Clausen, J.B. Bramsen, B. Finsen, C.K. Damgaard, J. Kjems, Natural RNA circles function as efficient microRNA sponges, Nature, 495
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16
ACCEPTED MANUSCRIPT
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(2013) 384-388. [33] T.B. Hansen, E.D. Wiklund, J.B. Bramsen, S.B. Villadsen, A.L. Statham, S.J. Clark, J. Kjems, miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA, EMBO J, 30 (2011) 4414-4422. [34] T.B. Hansen, J. Kjems, C.K. Damgaard, Circular RNA and miR-7 in cancer, Cancer Res, 73 (2013) 5609-5612. [35] Y.W. Jeske, J. Bowles, A. Greenfield, P. Koopman, Expression of a linear Sry transcript in the mouse genital ridge, Nat Genet, 10 (1995) 480-482. [36] A. Hacker, B. Capel, P. Goodfellow, R. Lovell-Badge, Expression of Sry, the mouse sex determining gene, Development, 121 (1995) 1603-1614. [37] M.W. Hentze, T. Preiss, Circular RNAs: splicing's enigma variations, EMBO J, 32 (2013) 923-925. [38] F. Li, L. Zhang, W. Li, J. Deng, J. Zheng, M. An, J. Lu, Y. Zhou, Circular RNA ITCH has inhibitory effect on ESCC by suppressing the Wnt/beta-catenin pathway, Oncotarget, 6 (2015) 6001-6013. [39] L.F. Thomas, P. Saetrom, Circular RNAs are depleted of polymorphisms at microRNA binding sites, Bioinformatics, 30 (2014) 2243-2246. [40] Q.F. Yin, L. Yang, Y. Zhang, J.F. Xiang, Y.W. Wu, G.G. Carmichael, L.L. Chen, Long noncoding RNAs with snoRNA ends, Mol Cell, 48 (2012) 219-230. [41] W.W. Du, W. Yang, E. Liu, Z. Yang, P. Dhaliwal, B.B. Yang, Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2, Nucleic Acids Res, 44 (2016) 2846-2858. [42] C.Y. Chen, P. Sarnow, Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs, Science, 268 (1995) 415-417. [43] W.R. Jeck, N.E. Sharpless, Detecting and characterizing circular RNAs, Nat Biotechnol, 32 (2014) 453-461. [44] A. Kos, R. Dijkema, A.C. Arnberg, P.H. van der Meide, H. Schellekens, The hepatitis delta (delta) virus possesses a circular RNA, Nature, 323 (1986) 558-560. [45] Y. Dou, D.J. Cha, J.L. Franklin, J.N. Higginbotham, D.K. Jeppesen, A.M. Weaver, N. Prasad, S. Levy, R.J. Coffey, J.G. Patton, B. Zhang, Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes, Sci Rep, 6 (2016) 37982. [46] I. Ahmed, T. Karedath, S.S. Andrews, I.K. Al-Azwani, Y.A. Mohamoud, D. Querleu, A. Rafii, J.A. Malek, Altered expression pattern of circular RNAs in primary and metastatic sites of epithelial ovarian carcinoma, Oncotarget, 7 (2016) 36366-36381. [47] J. Chen, Y. Li, Q. Zheng, C. Bao, J. He, B. Chen, D. Lyu, B. Zheng, Y. Xu, Z. Long, Y. Zhou, H. Zhu, Y. Wang, X. He, Y. Shi, S. Huang, Circular RNA profile identifies circPVT1 as a proliferative factor and prognostic marker in gastric cancer, Cancer Lett, 388 (2016) 208-219. [48] W. Xia, M. Qiu, R. Chen, S. Wang, X. Leng, J. Wang, Y. Xu, J. Hu, G. Dong, P.L. Xu, R. Yin, Circular RNA has_circ_0067934 is upregulated in esophageal squamous cell carcinoma and promoted proliferation, Sci Rep, 6 (2016) 35576. [49] P. Yang, Z. Qiu, Y. Jiang, L. Dong, W. Yang, C. Gu, G. Li, Y. Zhu, Silencing of
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cZNF292 circular RNA suppresses human glioma tube formation via the Wnt/beta-catenin signaling pathway, Oncotarget, 7 (2016) 63449-63455. [50] A. Bachmayr-Heyda, A.T. Reiner, K. Auer, N. Sukhbaatar, S. Aust, T. Bachleitner-Hofmann, I. Mesteri, T.W. Grunt, R. Zeillinger, D. Pils, Correlation of circular RNA abundance with proliferation--exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues, Sci Rep, 5 (2015) 8057. [51] J. Guarnerio, M. Bezzi, J.C. Jeong, S.V. Paffenholz, K. Berry, M.M. Naldini, F. Lo-Coco, Y. Tay, A.H. Beck, P.P. Pandolfi, Oncogenic Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal Translocations, Cell, 165 (2016) 289-302. [52] P. Li, S. Chen, H. Chen, X. Mo, T. Li, Y. Shao, B. Xiao, J. Guo, Using circular RNA as a novel type of biomarker in the screening of gastric cancer, Clin Chim Acta, 444 (2015) 132-136. [53] S.J. Conn, K.A. Pillman, J. Toubia, V.M. Conn, M. Salmanidis, C.A. Phillips, S. Roslan, A.W. Schreiber, P.A. Gregory, G.J. Goodall, The RNA binding protein quaking regulates formation of circRNAs, Cell, 160 (2015) 1125-1134. [54] S. Ghosal, S. Das, R. Sen, P. Basak, J. Chakrabarti, Circ2Traits: a comprehensive database for circular RNA potentially associated with disease and traits, Front Genet, 4 (2013) 283. [55] Y. Fang, J.L. Xue, Q. Shen, J. Chen, L. Tian, MicroRNA-7 inhibits tumor growth and metastasis by targeting the phosphoinositide 3-kinase/Akt pathway in hepatocellular carcinoma, Hepatology, 55 (2012) 1852-1862. [56] B. Kefas, J. Godlewski, L. Comeau, Y. Li, R. Abounader, M. Hawkinson, J. Lee, H. Fine, E.A. Chiocca, S. Lawler, B. Purow, microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma, Cancer Res, 68 (2008) 3566-3572. [57] S.D. Reddy, K. Ohshiro, S.K. Rayala, R. Kumar, MicroRNA-7, a homeobox D10 target, inhibits p21-activated kinase 1 and regulates its functions, Cancer Res, 68 (2008) 8195-8200. [58] O. Saydam, O. Senol, T. Wurdinger, A. Mizrak, G.B. Ozdener, A.O. Stemmer-Rachamimov, M. Yi, R.M. Stephens, A.M. Krichevsky, N. Saydam, G.J. Brenner, X.O. Breakefield, miRNA-7 attenuation in Schwannoma tumors stimulates growth by upregulating three oncogenic signaling pathways, Cancer Res, 71 (2011) 852-861. [59] R.J. Webster, K.M. Giles, K.J. Price, P.M. Zhang, J.S. Mattick, P.J. Leedman, Regulation of epidermal growth factor receptor signaling in human cancer cells by microRNA-7, J Biol Chem, 284 (2009) 5731-5741. [60] S. Xiong, Y. Zheng, P. Jiang, R. Liu, X. Liu, Y. Chu, MicroRNA-7 inhibits the growth of human non-small cell lung cancer A549 cells through targeting BCL-2, Int J Biol Sci, 7 (2011) 805-814. [61] H. Okuda, F. Xing, P.R. Pandey, S. Sharma, M. Watabe, S.K. Pai, Y.Y. Mo, M. Iiizumi-Gairani, S. Hirota, Y. Liu, K. Wu, R. Pochampally, K. Watabe, miR-7 suppresses brain metastasis of breast cancer stem-like cells by modulating KLF4, Cancer Res, 73 (2013) 1434-1444.
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[62] L. Jiang, X. Liu, Z. Chen, Y. Jin, C.E. Heidbreder, A. Kolokythas, A. Wang, Y. Dai, X. Zhou, MicroRNA-7 targets IGF1R (insulin-like growth factor 1 receptor) in tongue squamous cell carcinoma cells, Biochem J, 432 (2010) 199-205. [63] X. Zhao, W. Dou, L. He, S. Liang, J. Tie, C. Liu, T. Li, Y. Lu, P. Mo, Y. Shi, K. Wu, Y. Nie, D. Fan, MicroRNA-7 functions as an anti-metastatic microRNA in gastric cancer by targeting insulin-like growth factor-1 receptor, Oncogene, 32 (2013) 1363-1372. [64] X. Kong, G. Li, Y. Yuan, Y. He, X. Wu, W. Zhang, Z. Wu, T. Chen, W. Wu, P.E. Lobie, T. Zhu, MicroRNA-7 inhibits epithelial-to-mesenchymal transition and metastasis of breast cancer cells via targeting FAK expression, PLoS One, 7 (2012) e41523. [65] B. Wang, F. Sun, N. Dong, Z. Sun, Y. Diao, C. Zheng, J. Sun, Y. Yang, D. Jiang, MicroRNA-7 directly targets insulin-like growth factor 1 receptor to inhibit cellular growth and glucose metabolism in gliomas, Diagn Pathol, 9 (2014) 211. [66] N. Zhang, X. Li, C.W. Wu, Y. Dong, M. Cai, M.T. Mok, H. Wang, J. Chen, S.S. Ng, M. Chen, J.J. Sung, J. Yu, microRNA-7 is a novel inhibitor of YY1 contributing to colorectal tumorigenesis, Oncogene, 32 (2013) 5078-5088. [67] Y.T. Chou, H.H. Lin, Y.C. Lien, Y.H. Wang, C.F. Hong, Y.R. Kao, S.C. Lin, Y.C. Chang, S.Y. Lin, S.J. Chen, H.C. Chen, S.D. Yeh, C.W. Wu, EGFR promotes lung tumorigenesis by activating miR-7 through a Ras/ERK/Myc pathway that targets the Ets2 transcriptional repressor ERF, Cancer Res, 70 (2010) 8822-8831. [68] A.M. Cheng, M.W. Byrom, J. Shelton, L.P. Ford, Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis, Nucleic Acids Res, 33 (2005) 1290-1297. [69] Z. Yu, L. Ni, D. Chen, Q. Zhang, Z. Su, Y. Wang, W. Yu, X. Wu, J. Ye, S. Yang, Y. Lai, X. Li, Identification of miR-7 as an oncogene in renal cell carcinoma, J Mol Histol, 44 (2013) 669-677. [70] M.S. Ebert, J.R. Neilson, P.A. Sharp, MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells, Nat Methods, 4 (2007) 721-726. [71] M.S. Ebert, P.A. Sharp, MicroRNA sponges: progress and possibilities, RNA, 16 (2010) 2043-2050. [72] C.W. Lin, Y.L. Chang, Y.C. Chang, J.C. Lin, C.C. Chen, S.H. Pan, C.T. Wu, H.Y. Chen, S.C. Yang, T.M. Hong, P.C. Yang, MicroRNA-135b promotes lung cancer metastasis by regulating multiple targets in the Hippo pathway and LZTS1, Nat Commun, 4 (2013) 1877. [73] Y. Liu, Y. Han, H. Zhang, L. Nie, Z. Jiang, P. Fa, Y. Gui, Z. Cai, Synthetic miRNA-mowers targeting miR-183-96-182 cluster or miR-210 inhibit growth and migration and induce apoptosis in bladder cancer cells, PLoS One, 7 (2012) e52280.
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Figure 1. Biogenesis of circRNAs.
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Exon-skipping of pre-mRNA produces linear mRNAs and additional remnants form
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circles, or exonic circRNAs. Introns with complementary ALU repeats form circles
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through base-pairing to make ciRNA (circular intron RNAs). CircRNAs that contain
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both exons and introns are RIciRNA (Exon-Intron circRNA).
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Figure 2. Functions of circRNAs.
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(A) Acting as an miRNA sponge or ceRNA. (B) Directly targeting mRNA by partly
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base pairing. (C) Binding RNA binding protein (RBP) and AGO to regulate protein
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expression. (D) Used as the template of protein synthesis. IRES: Internal ribosome
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entry site.
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Figure 3. CDR1as /miR-7 and tumor.
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(A) CDR1as functions as an miR-7 buffer to inhibit the expression of miR-7’s targets.
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(B) The balance of CDR1as /miR-7 impacts a number of signaling pathways to
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regulate tumorigenesis, development and metastasis. EGFR: epidermal growth factor
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receptor; IRS-1/2: insulin receptor substrate-1/2; Pak1: p21-activated kinase-1; Ack1:
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activated CDC42 kinase 1; PIK3CD: phosphatidylinositol 3-kinase catalytic subunit δ;
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mTOR: mammalian target of rapamycin; IGF1R: insulin-like growth factor 1 receptor;
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KLF4: Kruppel-like factor 4; EMT: epithelial-to-mesenchymal transition; CSC:
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cancer stem cell.
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Highlights: 1. circRNA is exonic, intronic or retained-intronic. 2. CircRNAs mediate transcriptional and post-transcriptional regulation of gene and protein expression. 3. CircRNAs are critical for cancer biology.