1、COMMENTOpen AccessCircular RNAs as promising biomarkers incancer:detection,function,and beyondShengli Li1and Leng Han1,2*Editorial summaryCircular RNAs(circRNAs)are 35 covalently closed RNArings produced from back-splicing of precursor mRNAin eukaryotes.Recent studies,using both computationaland exp
2、erimental approaches,have allowed advancedcharacterization of circRNAs,leading the research fieldinto a new era and shedding light on the contributionof circRNAs to disease.Circularization diversifies the transcriptomeCircular RNAs(circRNAs)arise through out-of-ordersplicing,which involves covalent
3、ligation between thedownstream 5 splice sites and the upstream 3 splicesites of precursor messenger RNA(pre-mRNA;a processcalled back-splicing)1.In effect,the generation ofcircRNAs diversifies the eukaryotic transcriptome,in-creasing the functional capacity of a gene.circRNAs aregenerally expressed
4、at lower abundance levels than lin-ear transcripts,and until recent years,they were viewedas splicing noise or ligation artifacts.They can be circular-izedfrommanygenomicsources,includingexons(ecircRNA),introns(ciRNA),exon-introns(EIciRNA),orfusion transcripts(f-circRNA)of parental genes.circRNAexpr
5、ession is widespread among eukaryotic organisms,but importantly exhibits cell-specific and tissue-specificpatterns.Specific factors are involved in the regulation of cir-cRNA biogenesis 1,a process that involves the spliceoso-mal splicing mechanism wherein RNA-binding proteinsparticipate in the form
6、ation of circRNAs by binding toand probably stabilizing the back-splicing process.Thesplicing factor Quaking(QKI)has been demonstrated toregulate a variety of circRNAs that are involved in the The Author(s).2019 Open Access This article is distributed under the terms of the Creative Commons Attribut
7、ion 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 m
8、ade.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.*Correspondence:leng.hanuth.tmc.edu1Department of Biochemistry and Molecular Biology,McGovern MedicalSchool at The Univ
9、ersity of Texas Health Science Center at Houston,Houston,TX 77030,USA2Center for Precision Health,The University of Texas Health Science Center atHouston,Houston,TX 77030,USAepithelialmesenchymal transition(EMT)in humans,ex-emplifying a role for circRNA regulation in a biologicalprocess that could h
10、ave implications for diseases such ascancer 1.Further investigations of factors that directly orindirectly impact back-splicing are needed to enrich ourunderstanding of the biogenesis of circRNAs.circRNAs have been shown to function through spongingmicroRNAs,by interacting with proteins,by regulatin
11、g thetranscription of parental genes,or by encoding polypeptides1.For example,Cerebellar degeneration-related protein 1antisense RNA(CDR1as)participates in the regulation ofgenes that contain miR-7-binding sites through competitiveinteraction with miR-7,whereas circFOXO3 acts as anaccelerator of car
12、diac senescence by interacting with thetranscription factor E2F1 and the anti-senescent proteinID-1.circRNAs can also regulate the transcription of theirparental genes specifically.For example,circEIF3J has beenshown to enhance the expression of its parental gene byinteracting with U1 small nuclear
13、ribonucleoproteins andwith Pol II.CircZNF609 is an example of a circRNA thatcan be translated into a polypeptide,which may play afunctional role in regulating myoblast proliferation 1.Dysregulation of circRNA expression could lead toalterations in these processes,and there is increasingevidence of a
14、 role for circRNAs as regulatory RNA mol-ecules in tissue homeostasis and in human diseasessuch as cancer.Therefore,it is essential that circRNAsare detected and quantified accurately so that theirfunctions can be investigated further.Use of RNA deep-sequencing technology toidentify circRNAsThe adve
15、nt of high-throughput RNA deep-sequencingtechnology(RNA-seq)brought the encouraging discov-eries that,rather than being sequencing artifacts,cir-cRNAs are pervasively expressed in human genes 1and can be validated by quantitative PCR(qPCR)2.The choice of RNA library preparations before sequen-cing w
16、ill affect the detection of circRNAs 2.Currently,the most commonly used RNA-seq library preparationLi and Han Genome Medicine (2019)11:15 https:/doi.org/10.1186/s13073-019-0629-7strategies for circRNA detection are Ribo-Zero(ribosomeRNA(rRNA)depletion)and RNase R.Ribo-Zero librariesinclude both line
17、ar and circular RNAs after rRNA deple-tion,and thus do not provide tailored enrichment of cir-cRNAs.Their advantage is that they retain ample RNAinformation to facilitate downstream analysis.By contrast,the RNase R library digests linear RNA while the cova-lently closed loop structure of circRNAs al
18、lows them toelude exonucleolytic degradation,resulting in the en-richment of circRNAs.The digestion of linear RNAslimits the application of RNase R libraries in furtherdownstream analysis.In a recent study,Vo et al.3 employed exome captureRNA-seq to detect circRNAs.By targeting gene bodies,they achi
19、eved better enrichment for circRNA than that inthe Ribo-Zero libraries,while simultaneously preservinglinear RNAs.Thus,by achieving a balance between theenrichment of circRNAs and circular-to-linear ratios,theirprotocol complements conventional Ribo-Zero or RNaseR strategies for systematic investiga
20、tions of circRNAs.This protocol requires less than 5g of total RNA,sug-gesting that it will offer a significant advantage when usedfor clinical biospecimens that provide limited extractedRNA.Although the strategy is limited to circRNAs inknown exonic regions,and thus probably misses cir-cRNAs that o
21、riginate from intronic and intergenic re-gions,Vo et al.3 successfully characterized circRNAs inmore than 2000 tissue samples and 28 cell lines.They alsoidentified read-through circRNAs,a novel class of cir-cRNAs involving exons that originate from multiple genes.Furthermore,Vo et al.3 built a compr
22、ehensive catalogof circRNAs in human cancers,MiOncoCirc.This is amuch larger compendium than any other circRNA dataresource,including the Cancer-Specific CircRNA Data-base 4.By exploring MiOncoCirc,Vo et al.3 were ableto show a strong tissue-specific pattern of circRNAsacross different cancer types.
23、They also demonstrated thatcircRNAs that were identified in prostate cancer tissuesamples could be reliably detected in urine samples,sug-gesting the exciting possibility that circRNAs could havepotential for use as biomarkers in the noninvasive diagno-sis of human cancers.Thus,MiOncoCirc is a valua
24、bleresource that will promote the identification of novel cir-cRNAs as diagnostic and therapeutic targets.Computational approaches to detect circRNAsAlongside the burst of RNA-seq data,a variety of compu-tational algorithms for the identification and visualizationof circRNAs have recently been devel
25、oped 5.Most toolsare based on detecting back-splicing junctions(BSJs),which are junctions between sequences that occur inthe order opposite to that in the reference genome,indicating circularity 6.These approaches can be clas-sified as split-alignment-based approaches(i.e.,readsspanning BSJs are spl
26、it into segments and then alignedto a reference sequence using tools such as CIRCex-plorer,CIRI,and find_circ)or pseudo-reference-basedapproaches(i.e.,in which a pseudo-reference based onall possible BSJs is constructed and the reads arealigned against this pseudo-reference using tools suchas KNIFE,
27、NCLscan,and PTESFinder)6.Diverse cir-cRNA transcripts might be formed from a single paren-tal gene,however,and to date these algorithms havelimited power to detect and quantify the internal struc-tures of circRNAs accurately using the same BSJs.By con-sidering the internal components of circRNA,Zhen
28、g et al.7 proposed a new strategy,reverse overlap(RO),to re-construct full-length circRNAs.The CIRI-full algorithmcombines both RO and BSJ reads to allow quantificationof circRNAs at the isoform level and is better than existingmethods at detecting low-abundance circRNAs.Using thispowerful algorithm
29、,Zheng et al.7 were able to probe forlinks between disease and isoform specificity;for example,they observed an isoform switch in circZDBF2 from a447-nucleotide(nt)isoform in normal liver tissues to a334-nt isoform in liver cancer,providing a candidate forfuture functional and/or biomarker analysis.
30、CIRI-full pro-motes the accurate quantification,differential analysis,and alternative splicing analysis of circRNA transcriptsand will greatly improve our understanding of circRNA atup to isoform-level resolution.Functional characterization of circRNAsCurrent analyses indicate that perturbation of c
31、ircRNAs iswidespread in human cancers 1.One of the best-knowncircular RNAs,CDR1as,has been shown to promote(byacting as a sponge for miR-7)the upregulation of onco-genic factors(such as CCNE1 and PIK3CD)that aretargeted by miR-7,thus regulating the proliferation oftumor cells 1.Targeting these funct
32、ional circRNAs,forexample,by interfering with their biogenesis or their inter-actions with antisense oligonucleotides,might be a prom-ising therapeutic strategy for cancer 8.In a recent study,Chen et al.9 characterized circRNAs in prostate cancerpatients and demonstrated that altered circRNAs were a
33、s-sociated with prostate cancer progression.Interestingly,they showed that circRNA abundance was significantlyassociated with more read-through and fusion events,highlighting the potential link between fusion eventsand circRNA biogenesis.Remarkably,a genome-wideloss-of-function screen using small ha
34、irpin RNA todeplete circRNAs specifically revealed a total of 171 cir-cRNAs that were essential for cell proliferation in prostatecancer.These essential circRNAs showed functions thatwere distinct from those of their linear mRNA counter-parts;for example,circular casein kinase 1 gamma 3(circCSNK1G3)
35、promoted cell growth by interacting withmiR-181.This study also implies the contribution ofLi and Han Genome Medicine (2019)11:15 Page 2 of 3transcriptome diversity in human cancer by revealing thefunctional pathological significance of circRNAs 9.Future directions and therapeutic potentialDespite a
36、dvancements in the development of treatmentoptions for cancer,most cancer types continue to lackfully characterized and effective targeted therapies.Theidentification of circRNAs as targets for novel cancertherapies,as well as prognostic and diagnostic tools,rep-resents a promising frontier.In parti
37、cular,the stable cir-cular structure of circRNAs lengthens their half-life,especially in cell-free samples(such as blood and urine),creating potential for the use of circRNAs as biomarkersin patient samples from noninvasive sources.For ex-ample,abundant and stable circRNAs have been de-tected in hum
38、an blood exosomes and therefore holdpromise in the early diagnosis of cancers 10.Despite recent advances in characterizing circRNAs inhuman cancers,significant challenges remain becausedeveloping circRNA-targeted therapy will require a dee-per understanding of the molecular features,biogenesis,and f
39、unctional effects of circRNAs in cancer cells.Therefined detection methods used in the recent studies de-scribed above are leading the way into a new era of under-standing the features and functions of circRNAs,providinggreat opportunities to address the remaining challenges.Itis expected that the c
40、omputational methods and experi-mental systems established in cancer research will be ap-plicable to other diseases,thereby greatly leveraging theimpact of these approaches.AbbreviationsBSJ:Back-splicing junction;CDR1as:Cerebellar degeneration-related protein 1antisense RNA;circRNA:Circular RNA;RNA-
41、seq:High-throughput RNA deep-sequencing technologyAcknowledgementsWe thank LeeAnn Chastain for editorial assistance.FundingThis work was supported by the Cancer Prevention&Research Institute ofTexas(award RR150085 to LH).Authors contributionsSL and LH jointly wrote the article and approved the final
42、 manuscript.Bothauthors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.References1.Li X,Yang L,Chen
43、 LL.The biogenesis,functions,and challenges of circularRNAs.Mol Cell.2018;71:42842.2.Szabo L,Salzman J.Detecting circular RNAs:bioinformatic and experimentalchallenges.Nat Rev Genet.2016;17:67992.3.Vo JN,Cieslik M,Zhang Y,Shukla S,Xiao L,Zhang Y,et al.The landscape ofcircular RNA in cancer.Cell.2019
44、;176:86981.4.Xia S,Feng J,Chen K,Ma Y,Gong J,Cai F,et al.CSCD:a database forcancer-specific circular RNAs.Nucleic Acids Res.2018;46:D9259.5.Xiang Y,Ye Y,Zhang Z,Han L.Maximizing the utility of cancertranscriptomic data.Trends Cancer.2018;4:82337.6.Gao Y,Zhao F.Computational strategies for exploring
45、circular RNAs.TrendsGenet.2018;34:389400.7.Zheng Y,Ji P,Chen S,Hou L,Zhao F.Reconstruction of full-length circularRNAs enables isoform-level quantification.Genome Med.2019;11:2.8.Kristensen LS,Hansen TB,Ven MT,Kjems J.Circular RNAs in cancer:opportunities and challenges in the field.Oncogene.2018;37
46、:55565.9.Chen S,Huang V,Xu X,Livingstone J,Soares F,Jeon J,et al.Widespreadand functional RNA circularization in localized prostate cancer.Cell.2019;176:83143.10.Li Y,Zheng Q,Bao C,Li S,Guo W,Zhao J,et al.Circular RNA is enrichedand stable in exosomes:a promising biomarker for cancer diagnosis.CellRes.2015;25:9814.Li and Han Genome Medicine (2019)11:15 Page 3 of 3
