Circular RNAs... human cancer_Fan Hu.pdf

Cancer Biol Med 2023.doi:10.20892/j.issn.2095-3941.2022.0466REVIEWCircular RNAs:implications of signaling pathways and bioinformatics in human cancerFan Hu*,Yin Peng*,Xinmin Fan,Xiaojing Zhang,Zhe JinGuangdong Provincial Key Laboratory of Genome Stability and Disease Prevention and Regional Immunity and Diseases,Department of Pathology,School of Basic Medical Sciences,Medical School,Shenzhen University,Shenzhen 518060,ChinaABSTRACT Circular RNAs(circRNAs)form a class of endogenous single-stranded RNA transcripts that are widely expressed in eukaryotic cells.These RNAs mediate post-transcriptional control of gene expression and have multiple functions in biological processes,such as transcriptional regulation and splicing.They serve predominantly as microRNA sponges,RNA-binding proteins,and templates for translation.More importantly,circRNAs are involved in cancer progression,and may serve as promising biomarkers for tumor diagnosis and therapy.Although traditional experimental methods are usually time-consuming and laborious,substantial progress has been made in exploring potential circRNA-disease associations by using computational models,summarized signaling pathway data,and other databases.Here,we review the biological characteristics and functions of circRNAs,including their roles in cancer.Specifically,we focus on the signaling pathways associated with carcinogenesis,and the status of circRNA-associated bioinformatics databases.Finally,we explore the potential roles of circRNAs as prognostic biomarkers in cancer.KEYWORDS circRNA;cancer;signaling pathway;database;bioinformaticsIntroductionCircular RNAs(circRNAs)are an emerging class of endoge-nous RNAs abundantly expressed in eukaryotic cells.These molecules are generated from precursor mRNAs through non-canonical splicing and are widely expressed in diverse species.circRNAs include exonic circRNAs,exon-intron cir-cRNAs(EIciRNAs),and circular intronic RNAs(ciRNAs)1,2.circRNAs function mainly as microRNA(miR)sponges and RNA-binding protein(RBP)scaffolds,and they encode novel proteins that regulate gene transcription or protein transla-tion3.Recent studies have indicated that circRNAs are involved in miR inhibition,epithelial-mesenchymal transition(EMT),and tumorigenesis.Furthermore,circRNAs expression can be tissue specific,and evidence indicates that some circRNAs are translated4.In contrast to linear RNAs,circRNAs form a covalently closed loop structure without a 5 cap or 3 tail,and have much longer half-lives5,6.Advances in high-throughput sequencing technology and novel bioinformatics algorithms have facilitated the systematic detection of circRNAs,most of which are stable,abundant,and conserved,and show an incredible diversity of tissue-specific expression.Studies have indicated that circRNAs are associated with many clinical characteristics and thus may provide important guidance for the accurate diagnosis and treatment of cancer7.Signaling pathways play key roles in carcinogenesis.For example,the Wnt pathway is an evolutionarily conserved pathway8,9 that is divided into 3 classes:Wnt/-catenin sig-naling,Wnt/planar cell polarity signaling,and Wnt/Ca sign-aling.Wnt/-catenin signaling plays critical roles in embry-onic development,tissue renewal,and regeneration10,and is significantly correlated with several types of cancers,such as lung cancer11,gastric cancer(GC)12,colorectal cancer(CRC)13,bladder cancer14,glioma15,and chronic lymphocytic leukemia16.Similarly,aberrant activation of the other signa-ling pathways has been found to significantly correlate with various cancers.Accumulating evidence indicates that circR-NAs are associated with various cancer processes,including*These authors contributed equally to this work.Correspondence to:Zhe Jin and Xiaojing ZhangE-mail: and ORCID ID:https:/orcid.org/0000-0001-7911-7970 and https:/orcid.org/0000-0002-0595-3500Received August 4,2022;accepted December 19,2022Available at www.cancerbiomed.org2023 Cancer Biology&Medicine.Creative Commons Attribution-NonCommercial 4.0 International LicenseCancer Biol Med Vol 20,No 2 February 2023 105cancer initiation,progression,and metastasis,via signaling pathways17.Traditional experimental approaches have been impor-tant in exploring the biological functions and characteris-tics of molecules as well as cancer pathogenesis.However,these methods can be time-consuming and laborious.With the discovery of large numbers of circRNAs,an urgent need exists to use in silico methods to reveal their characteristics,and guide the rational design of expensive and laborious clinical trials2.In this review,we summarize current understanding of the biological characteristics and functions of circRNAs,with a focus on signaling pathways associated with carcinogenesis.This information should provide insights into potential new targets for the treatment of cancers.Finally,we discuss the cur-rent status of circRNA bioinformatics databases,and explore the potential roles of circRNAs as prognostic biomarkers and therapeutic targets in cancer.RNA circularization and circRNA biogenesiscircRNAs are derived from pre-messenger RNAs(pre-mR-NAs)and originate from exons,introns,antisense RNAs,and intergenic regions.Under non-pathological conditions,circR-NAs control gene expression by regulating gene transcription,RNA splicing,and scaffold assembly18,19.In addition,some circRNAs encode functional peptides.Four mechanisms of circularization have been confirmed:intron base-pairing-driven circularization(Figure 1A),RBP-driven circulariza-tion(Figure 1B),GU/C-rich sequence-driven circularization(Figure 1C),and pre-tRNA-mediated generation of tRNA intronic circular RNA(tricRNA)(Figure 1D)2.Recent stud-ies have revealed that chromosomal translocations lead to the generation of fused circRNA20.The most common types of circRNA are ciRNAs,EIciRNAs,and exonic circRNAs,which account for the largest proportion(85%).EcircRNAs are distributed mainly in the cytoplasm,whereas ciRNAs and EIciRNAs exist mainly in the nucleus21,22.In eukaryotes,pre-mRNAs are generally processed to gen-erate linear mRNAs through canonical splicing,whereas circRNAs are formed through alternative“head-to-tail”back-splicing events20,in a process involving the formation of a covalently closed loop through reverse ligation of a down-stream-splice donor site to an upstream-splice acceptor site.circRNA circularization is promoted by RBP-mediated bridg-ing of relevant intronic sequences in RNA1,20,22.Studies on circRNA biogenesis have expanded the understanding of the complexity of RNA transcriptional regulation;however,the mechanism of the back-splicing events that generate circRNAs remains to be fully elucidated.Potential functions of circRNAs in cancerInteractions with proteins as regulators and scaffoldsTranscription and splicingSome circRNAs interact with RNA polymerase II(Pol II),and consequently regulate the transcription and splicing of paren-tal genes.In the nucleolus,EIciRNAs and ciRNAs enhance parental gene transcription by interacting with the U1 small nuclear ribonucleoprotein or binding the Pol II promoter5.Similarly,ci-ankrd52 and ci-sirt7 localize to and interact with the elongating Pol II complex.Depletion of these ciR-NAs decreases the transcription levels of the ankyrin repeat domain 52(ANKRD52)or sirtuin 7(SIRT7)genes23.Circ-DNMT1 promotes the nuclear translation of p53 and acts on AU-rich element RNA-binding protein 1(AUF1),thereby resulting in cellular autophagy and target Dnmt1 mRNA sta-bility in breast cancer24.Thus,circRNAs compete with spliced pre-mRNAs by triggering transcription,and consequently balance the levels of circRNAs and corresponding mRNAs(Figure 1E).Protein recruitment and scaffoldingSome circRNAs serve as scaffolds that promote protein recruitment and assembly.A recent report has demonstrated that circndufb2 functions as a scaffold that binds the IGF2BP proteins with TRIM25,a positive regulator of tumor progres-sion and metastasis in non-small cell lung cancer(NSCLC)25.The circRNA FECR1,which is generated from the Friend leu-kemia virus integration 1(FLI-1)oncogene,recruits TET1 to the promoter of FLI-1 and promotes breast cancer metasta-sis26.Some circRNAs also facilitate reaction kinetics by bind-ing enzymes and substrates.Circ-Foxo3 halts cell cycle pro-gression by forming a ternary complex with cyclin-dependent kinase 2 and its inhibitor p2127.Thus,circRNAs can act as scaf-folds that mediate complex formation by specific enzymes and substrates involved in carcinogenesis(Figure 1F).106 Hu et al.The hallmarks of circRNA in human cancerPre-mRNAA.Intron base-pairing-driven circularizationRBPRBPSDSAAluB.RBPs-drivenD.Pre-tRNApre-mRNACompetitioncircRNAcircRNAcircRNAcircRNASubstrateRelated signalingpathwayGene expressionEnzymecircRNA-aaLinearRNALinearRNA-aacircRNA genesBHBmotifttricRNASpliceosomeElciRNAecircRNA ciRNAgenerates tricRNAC.GU/C-rich sequence-driven circularizationH.M6A/IRES driventranslationYTHDF3m6AE.Enhancing transcriptionand splicingPolIIF.Protein recruitmentand scaffoldG.miRNA/protein spongeC-rich elementGU-rich elementNucleuscircRNAmiRNAA G ORBPInteracting withhost geneCytoplasmTarget genesmiRNA mRNARelated signallingpathwayTarget proteinincreaseI.circRNAs in exosomesExo-circRNAcircularizationCompetitive withcanonical productProteinIRES40S40S43S60SAUGFigure 1 Biogenesis,mechanisms,and functions of circRNAs in cancer.(A)Intron base-pairing-driven circularization.(B)RBP-driven circu-larization.Looping of the introns(containing the splice donor site and splice acceptor site)flanked by exons is required for back-splicing.This looping can be facilitated by base-paring of complementary sequences between inverse-repeat Alu elements(A)or by RBP dimerization(B).RBPs bind intron-flanking introns and promote circularization of the pre-mRNA(or lariat),thus generating circRNAs.(C)GU/C-rich sequence-driven circularization.Pre-mRNAs comprising a 7-nucleotide(nt)GU-rich element and an 11-nt C-rich element consensus motif facilitate the generation of circRNAs.(D)Pre-tRNAs generate tricRNAs.An intron-containing pre-tRNA is cleaved by the tRNA spicing endonuclease(TSEN)complex,thus generating a tricRNA at the bulge-helix-bulge(BHB)motif;the intron termini then ligate and form a tricRNA.(E)circRNAs interact with Pol II,thereby regulating parental gene transcription and splicing.Competition between linear splicing and back-splicing of the pre-mRNA influences the balance between the 2 types of splicing.(F)circRNAs interact with proteins in several ways.circRNAs act as scaffolds that facilitate interactions between enzymes with their substrates.circRNAs can also recruit proteins to specific loci and promote protein assembly.(G)circRNAs function as miR and protein sponges.circRNAs containing miR response elements(MREs)can regulate miR-target mRNA expression through miR sequestration(or“sponging”).A highly expressed circRNA with many MREs is likely to function as an miR sponge and to positively regulate target mRNA translation.circRNAs containing binding motifs for RBPs might sponge these proteins and regulate their functions.(H)m6A and IRES-driven circRNA translation.A subset of circRNAs containing IRES and/or m6A modifications can serve as templates for translation and give rise to circRNA-specific peptides with the ORF crossing the back-splicing junctions.(I)circRNA transport via exosomes.circRNAs can be loaded into exosomes,thereby forming exo-circRNAs,which function as messengers in intercellular communication through the horizontal transfer of their cargo molecules to recipient cells.Cancer Biol Med Vol 20,No 2 February 2023 107miRs and protein spongescircRNAs inhibit the functions of miRs by competitive bind-ing or“sponging”through the formation of stable comple-mentary interactions.The circRNA-miR-mRNA axis is also involved in various cancer-associated pathways with both ago-nistic and antagonistic effects on carcinogenesis.CIRS-7,the first circRNA identified as an miR sponge,inhibits circRNA-7,through more than 70 conventional miR-7 binding sites,and is associated with cancer progression28,29.Moreover,circRUNX1 promotes papillary thyroid cancer(PTC)progression and metastasis by sponging miR-296-3p and regulating DDHD2 expression30.The circRNA UBE2Q2 promotes malignant pro-gression of GC by regulating the miR-370-3p/STAT3 axis31.CircORC5 suppresses GC progression by sponging miR-30c-2-3p and regulating AKT1S132.However,the miR sponge model is becoming increasingly controversial,because most circRNAs do not show strong sponging effects on miR-bind-ing sites33.Furthermore,the circRNA-miR axis regulates the activity of the corresponding linear mRNA.Thus,the mech-anisms through which circRNAs act as miR sponges,and consequently regulate gene and protein expression,require further investigation.In another model,the biological func-tion of circRNAs depends on interactions with RBPs,which have various roles in circRNA splicing,processing,folding,stabilization,and localization4,34.RBPs interact with circRNAs and form RNA-protein complexes,which in turn regulate the circularization of circRNAs,such as SCD-circRNA and circP-CNX,in cancers35,36.RBPs also interact with circRNAs,and hide or expose certain regions in a process essential for the correct splicing,localization,and translation of cellular com-ponents34.Thus,sponging of circRNAs by miRs or proteins is a key mechanism through which circRNAs perform multiple functions in cancer(Figure 1G).circRNA translationcircRNAs initially lack a 5-cap and 3-tail,and are therefore classified as non-coding RNAs;however,a small fraction of circRNAs(1%)are translated into functional proteins or micropeptides via one of 2 mechanisms:N6-methyladenosine(m6A)-driven or internal ribosome entry site(IRES)-driven translation.The m6A motif in the 5-untranslated region(5-UTR)is a major mechanism37-39(Figure 1H).Yang et al.39 have shown that m6A directly recruits the initiation fac-tor eIF4G2 for formation of the 43S complex,which in turn promotes the initiation of circRNA translation in human cells.IRES elements within the 5-UTRs of the upstream open reading frames(ORFs)function as RNA regulatory elements that initiate circRNA translation independently of the 5 cap structure40-48.Xia et al.41 have suggested that circ-AKT3 is generated by the circularization of exons 37 of AKT3,which contain both an ORF and an IRES sequence,and have specu-lated that the 5-UTR of circ-AKT3 is structurally folded into the IRES and encodes the AKT3-174 aa protein.Interestingly,circ-EGFR forms a polymetric novel protein complex known as rolling-translated EGFR48.We believe that this model pro-vides a new understanding of circRNAs that blurs the defini-tion of non-coding RNAs.Thus,circRNA-encoded peptides stand to become a new resource for anti-tumor protein drug screening of early tumor biomarkers,precise therapeutics,and molecules to aid in prognostication.Exosome-circRNAs(exo-circRNAs)in cancerExosomes are smal