Emerging function and potential diagnostic val.pdf

REVIEWOpen AccessEmerging function and potential diagnosticvalue of circular RNAs in cancerXianglun Cui1,Jianxun Wang2,Zongjun Guo3,Mengyang Li2,Mingyu Li1,Si Liu1,Haoran Liu1,Wenjing Li1,Xunhua Yin1,Jiaping Tao1and Wenhua Xu1*AbstractAs a novel class of endogenous RNAs,circRNAs,have a covalently closed continuous loop,with neither a 5to3polarity,nor a polyadenylated tail.Numerous circRNAs have been characterized by abundance,stabilization,conservation,and exhibit tissue/developmental stage-specific expression.Furthermore,circRNAs play vital roles intumorigenesis and metastasis,such as functioning as a ceRNA or miRNA sponge,interacting with protein andencoding protein.Increasing evidence has revealed that it potentially serves as a required novel biomarker forcancer diagnosis.This review summarized the latest research on circRNAs,including its classification and biogenesis,mechanism and functions,as well as circRNAs in different cancers,as a potential biomarker.Keywords:Circular RNA,Cancer,Biogenesis,Function,BiomarkerBackgroundAs a novel class of long non-coding RNAs,circularRNAs(circRNAs)are widely expressed in the tree of life13.circRNAs have originally been considered asnon-functional accidental by-products of aberrant spli-cing 4,which has not received enough attention.Withthe emergence of next-generation sequencing,especiallyRNA sequencing technology,numerous circRNAs havebeen found to be extensively expressed in eukaryoticcells.circRNAs are single-stranded transcripts derivedfrom exons,introns,or intergenic regions that have acovalently closed continuous loop without a polyadeny-lated tail 5.Due to the closed structure,circRNAs havebeen shown to be highly stable.Numerous circRNAsdisplay evolutionary conservation,and the expressionprofiles are cell type-or developmental stage-specific.Cancer is one of the most serious and life-threateningdiseases,which has high morbidity and mortality world-wide,and a high frequency of metastasis and recurrence.Hence,there is an urgent need to identify potential bio-markers for prognosis predication,and determine newtargets to design more powerful therapeutic approaches.Various studies have suggested that circRNAs are ofgreat significance in tumorigenesis and metastasis,suchas lung cancer 6,7,colorectal cancer 8,9,gastric can-cer 10,11,hepatocellular carcinoma(HCC)1215,breast cancer 16,17,and so on.The present studysummarized the latest research on circRNAs,includingits classification and biogenesis,mechanism and func-tions,as well as circRNAs in different cancers,as a po-tential biomarker.Classification and biogenesis of circRNAsAccording to composition,circRNAs can be classifiedinto three categories:(a)ecircRNAs contain only exonsequences with 35-linked,which account for over80%of discovered circRNAs 1820;(b)ciRNAs onlyconsist of intron sequences with 2 5-linked introniclariats,which are located in the nucleus 21,22;(c)EIciRNAs comprise of both exon and intron sequenceswith 3 5-linked,which are nuclear localized 23.There is another principle of classification based onbreakpoint location.According to location relationshipof circRNAs with adjacent coding RNA,they are classi-fied into five types:exonic,intronic,antisense,senseoverlapping and intergenic.The first two kinds,likeecircRNAs and ciRNAs,composed of introns and exons.Antisense:derived from the opposite strand,whose se-quences overlap with the linear mRNA.Sense overlap-ping:composed of same sequences as the linear mRNA,*Correspondence:1Department of Inspection,The medical faculty of Qingdao University,Qingdao 266003,ChinaFull list of author information is available at the end of the article The Author(s).2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License(http:/creativecommons.org/licenses/by/4.0/),which permits unrestricted use,distribution,andreproduction in any medium,provided you give appropriate credit to the original author(s)and the source,provide a link tothe Creative Commons license,and indicate if changes were made.The Creative Commons Public Domain Dedication waiver(http:/creativecommons.org/publicdomain/zero/1.0/)applies to the data made available in this article,unless otherwise stated.Cui et al.Molecular Cancer (2018)17:123 https:/doi.org/10.1186/s12943-018-0877-ybut not classified into exonic or intronic.Intergenic:consists of sequences located in noncoding region 24.After being synthesized by RNA polymerase II,the pre-cursor mRNAs(pre-mRNAs)are spliced and the intronsare removed,alternatively joining the exons to generatelinear mRNAs 25.CircRNAs are also generated frompre-mRNAs through different mechanisms.There arethree biogenesis mechanisms described below(Fig.1).Spliceosome-dependent lariat-driven circularizationExon circulation is spliceosome(or at least U1)-dependentas revealed by mutation of the 5 splice sites 26.In thismodel,the spliceosomes are assembled at back-splicingsite to promote the joining of the downstream 5donorsites with the upstream 3acceptor sites.The lariat is sub-sequently processed by internal splicing,which finally re-sults in the release of ecircRNAs or EIciRNAs.CircRNAsbiogenesis and canonical splicing compete with each other26.Besides,the efficiency of backsplicing is much lowerthan that of linear splicing 27.Backsplicing may occurpost-transcriptionally,in which the circulation is anexon-containing lariat produced by exon skipping 18.Inaddition,backsplicing can also occur co-transcriptionally,and the circular production from nascent mRNA does notneed a polyadenylation signal 28.The backsplicing in-volves single exon 18,19 or several exons 20 with inter-vening introns.Intron is nucleotide sequence between exons that is re-moved by RNA splicing during maturation of mRNA.However,some introns containing special motif escapefrom identification of debranching enzyme and formintron-derived ciRNAs.The essential motif consist of 7-ntGU-rich element located near the 5splice site,and 11-ntC-rich element near the branch point 29.As special ciR-NAs,tricRNAs are derived from pre-tRNAs splicing andconsist of intronic sequences.The biogenesis of tricRNAsconservatively exists in both archaea and eukaryotes reliesonthesplicingendonucleasecomplex,whichcanrecognize the bulge-helix-bulge(BHB)sequence motifand cleave pre-tRNAs.Subsequently,exon termini linkwith each other to form a mature tRNA,and intron ter-mini are ligated together to form tricRNA 30.Intron-pairing circulationThe pairing between two introns that flank the circular-izedexons,whichhaveacomplementaryinvertedabcdFig.1 Biogenesis of circRNAs.a circRNAs formation can occur through RBPs mediated folding of the pre-miRNA.b the pairing between the two intronsflanking the circularized exons,which have a complementary inverted sequence,can promote the backsplicing proceed in cis.c the back-splicing sitepromotes the joining of the downstream 5donor sites with the upstream 3acceptor sites.The lariat is subsequently processed by internal splicing,whichfinally results in the release of ecircRNAs or EIciRNAs.d tricRNA exon termini link with each other to form a mature tRNA,and intron termini are ligatedtogether to form tricRNACui et al.Molecular Cancer (2018)17:123 Page 2 of 14sequence,can promote the backsplicing proceed incis 31.The paratactic intronic structure makes thesplice donor close to the splice acceptor,and facili-tates the nucleophilic attack and cleavage.One of thecomplementary repeats is Alu elements 32,whichexist in more than 10%of the human genome.Aluelements derived from introns that flank circularizedexons are more likely to complement,compared toother origins.Besides,complementary Alu elementsare six-fold more likely present within flanking intronof circularized exons 1.The competition betweendifferent-locatedreversecomplementarysequencesleads to production of diverse circRNA isoforms froma single gene.Furthermore,RNA pairing can occur atnon-repetitive complementary sequences 31.It hasbeen reported that flanking sequence or structuralcomplementarity is absent in Drosophila RNA circula-tion.Similarly,only a small proportion of circRNAspossess flanking intronic complementary sequences inrice.Moreover,complementarysequences notlessthan 3040 nucleotides are able to assist circRNAbiogenesis 33.RNA-binding proteins(RBP)-induced circulationcircRNA formation can occur through RBPs mediatedfolding of the pre-miRNA.RBPs,including Muscleblind(MBL)26,Quaking(QKI)34,Fused-in sarcoma(FUS)35,are able to increase the rate of circulation bybridging relevant intronic sequences.The dimerizationof RBPs,which binding with up-and downstream of thecircularized exon,brings the 3 and 5 end of the circu-larized exons into close proximity and promotes theirsplicing.The flanking introns of circMBL contain con-served MBL binding sites.Moreover,the MBL interactswith its own pre-mRNA and stimulates cognate circRNAproduction.Conversely,mutation of the MBL bindingsites evidently reduces circMBL production 26.Regu-lated during epithelial-mesenchymal transition,QKI dy-namicallymodulatestheproductionofmorethanone-third of abundant circRNAs.Moreover,the RNA-and DNA-binding protein FUS binds to circularizingexon-intron junctions,and it regulates the production of136 circRNAs in in vitro-derived mouse motor neurons.In the contrary,there are two RBPs:ADAR1 and DHX9.As a negative regulation factor,these reduce the forma-tionofcircRNAs.Furthermore,double-strandedRNA-specific adenosine deaminase(ADAR)has beenfound to diminish circRNA expression through theadenosine-to-inosine(A-to-I)editingactivity,whichmakes RNA pairs anneal and reduces complementarityand backsplicing 32,36.Moreover,the nuclear RNAhelicase DHX9 can interact with inverted-repeat Alu ele-ments,downregulating Alu elements-induced intronpairing.Mechanism and function of circRNAs in cancerRecent studies indicate that circRNAs play a vital role inphysiologicalandpathologicalprocessesatthepost-transcription or transcription level.Here,we sum-marized the function and mechanism of circRNAs incancer(Fig.2).As competing endogenous RNAs or miRNA spongesCompeting endogenous RNA(ceRNA)is described as acomplex post-transcriptional regulatory network medi-ated by sequestrating miRNAs 37,38.The hypothesisshows that miRNA activity can be reduced by transcriptscontaining miRNA response elements(MREs),subse-quently upregulating miRNA target expression.Apartfrom mRNA,transcribed pseudogenes 38 and longnoncoding RNA(lncRNA)39,numerous studies havefound that many circRNAs regulate miRNA network asceRNAs 2,40.Furthermore,it has been shown that themajority of circRNAs are principally localized in thecytoplasm,suggesting that circRNAs may function as amiRNA sponge to sequestrate miRNAs(Fig.2a).Thereare two characterized circRNAs,CiRS-7 and circSRY,ver-ifiedthishypothesis.CiRS-7(circRNAspongeformiR-7)contains more than 60 conserved miR-7 targetsites,which are predominantly expressed in human andmouse brain 2,.CircSRY contains 16 putative miR-138target sites that functions as miR-138 sponges 41.Various circRNAs function as miRNAs sponges intumorigenesis and progression.Hsa_circ_0012673 func-tions as a miRNA sponge of miR-22,which targetserb-b2 receptor tyrosine kinase 3(ErbB3),promotinglung adenocarcinoma(LAC)cell proliferation 42.As atumor suppressor,circLARP4 is downregulated in gastriccancer(GC)tissues,suppressing gastric tumorigenesisand progression by sponging miR-424 and increasingLATS1 expression 10.WJ Huang et al.demonstratedthat hsa_circ_0000977 interacts with has-miR-874-3p,and subsequently promotes the expression of PLK1 inpancreatic ductal adenocarcinoma cancer 43.In hepa-tocellular carcinoma,circHIPK3 functions as a miRNAsponge of miR-124,which sequentially upregulates theexpression of AQP3,and promotes cell proliferation andmigration 12.Both circGFRA1 and GFRA1 are upregu-lated in triple negative breast cancer(TNBC),and cir-cGFRA1 functions as a ceRNA to regulate GFRA1expression by decoying miR-34a 16.Chengdi Yangobservedthatcirc-ITCHsuppressedtheaggressivebiologicalbehaviorsofbladdercancer(BCa),andupregulated the expression of p21 and PTEN throughdecoys miR-17 and miR-224 44.The expression ofcirc-SHKBP1 is elevated in glioma-exposed endothelialcells(GECs),which functions as a ceRNA via themiR-544a/FOXP1 or miR-379/FOXP2 pathway 45.Fur-thermore,L Chen et al.discovered that circRNA_100290Cui et al.Molecular Cancer (2018)17:123 Page 3 of 14was elevated in OSCC tissues,which upregulates CDK6expression through decoying miR-29b family members,playing a crucial role in OSCC progression such as tumorinvasion and metastasis 46.Upregulated circUBPA2 pro-motes osteosarcoma growth and inhibits apoptosis bydownregulating the expression of miR-143,consequentlyraising the expression of anti-apoptosis Bcl-2 47.Protein translationAlthough defined as a subclass of non-coding RNAs,in-creasing evidence have demonstrated that circRNAs havepotential to participate in translation 48,49(Fig.2b).Itcan be presumed that a protein-translated circRNA whensome of the following features are present:(A)circRNAshave ORF with sufficient length,which is essential for lin-ear mRNA protein translation;(B)it has the spanningbacksplicing junction ORF;(C)some of the necessaryregulation elements are present to the translation initi-ation upstream ORF,such as the internal ribosome entrysite(IRES)element,and the N6-methyladenosine(m6A)modifications near the start codon 50.Recently,at least two cases offer important evidencefor the existence of the translation of endogenouscircRNA-encoded peptides:circFXBW7 and cricSHPRH.These are of great significance in tumorigenesis and pro-gression.Y Yang et al.revealed that circFXBW7 canencode a novel 21-KDa protein,which was named,FBXW7-185aa.A dual luciferase vector system con-structed with full length or truncated putative circ-FBXW7IRES sequences was used to test for IRES activity.The re-sult revealed that only the full length circFBXW7 IRESgroup can induce the highest Luc/Ruc activity.Next,thecirc-FXBW7 vector and other control vectors were trans-fected into human cells,and FBXW-185aa was detected byaspecificantibodyandliquidchromatographytandem-massspectrometry.Theresultsuggeststhatcirc-FBXW7 has the potential to encode a novel protein.The protein FBXW7-185aa functions as a tumor suppres-sor by competitively binding with USP28,and preventingUSP28bindingtoFBXW7,subsequentlyinhibitingUSP28-inducedc-mycstabilization.Circ-FBXW7andFBXW7-185aa were downregulated in glioblastoma,andabcFig.2 Functions of circRNAs.a miRNA sponge circRNAs can reduce miRNA activity by transcripts containing MREs,subsequently upregulatingmiRNA target expression.b CircRNAs-Protein interaction The interaction between circRNAs and proteins can facilitate the interaction of multipleproteins.c Protein translation circRNAs containing ORF and IRES have the potential to participant in translationCui et al.Molecular Cancer (2018)17:123 Page 4 of 14suppress proliferation and ce