Circular RNAs pp 357-370 | Cite as

Prospective Advances in Circular RNA Investigation

  • Siti Aishah Sulaiman
  • Nor Azian Abdul MuradEmail author
  • Ezanee Azlina Mohamad Hanif
  • Nadiah AbuEmail author
  • Rahman Jamal
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1087)


circRNAs have emerged as one of the key regulators in many cellular mechanisms and pathogenesis of diseases. However, with the limited knowledge and current technologies for circRNA investigations, there are several challenges that need to be addressed for. These include challenges in understanding the regulation of circRNA biogenesis, experimental designs, and sample preparations to characterize the circRNAs in diseases as well as the bioinformatics pipelines and algorithms. In this chapter, we discussed the above challenges and possible strategies to overcome those limitations. We also addressed the differences between the existing applications and technologies to study the circRNAs in diseases. By addressing these challenges, further understanding of circRNAs roles and regulations as well as the discovery of novel circRNAs could be achieved.


circRNA Challenges Identification and analysis Future directions 


  1. 1.
    Haque S, Harries LW (2017) Circular RNAs (CircRNAs) in health and disease. Genes 8(12):1–17Google Scholar
  2. 2.
    Jeck WR, Sharpless NE (2014) Detecting and characterizing circular RNAs. Nat Biotechnol 32(5):453–461CrossRefGoogle Scholar
  3. 3.
    Pamudurti NR, Bartok O, Jens M et al (2017) Translation of CircRNAs. Mol Cell 66(1):9–21 e27CrossRefGoogle Scholar
  4. 4.
    Qu S, Liu Z, Yang X et al (2018) The emerging functions and roles of circular RNAs in cancer. Cancer Lett 414(2018):301–309CrossRefGoogle Scholar
  5. 5.
    Tay Y, Rinn J, Pandolfi PP (2014) The multilayered complexity of ceRNA crosstalk and competition. Nature 505(7483):344–352CrossRefGoogle Scholar
  6. 6.
    Lee SM, Kong HG, Ryu CM (2017) Are circular RNAs new kids on the block? Trends Plant Sci 22(5):357–360CrossRefGoogle Scholar
  7. 7.
    Liu T, Zhang L, Chen G et al (2017) Identifying and characterizing the circular RNAs during the lifespan of Arabidopsis leaves. Front Plant Sci 8(July):1–9Google Scholar
  8. 8.
    Dou Y, Cha DJ, Franklin JL et al (2016) Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes. Sci Rep 6(October 2015):1–11Google Scholar
  9. 9.
    Meng S, Zhou H, Feng Z et al (2017) CircRNA: functions and properties of a novel potential biomarker for cancer. Mol Cancer 16(1):1–8CrossRefGoogle Scholar
  10. 10.
    Wang Y, Wang Z (2015) Efficient backsplicing produces translatable circular mRNAs. RNA 21(2):172–179CrossRefGoogle Scholar
  11. 11.
    Zhong Z, Lv M, Chen J (2016) Screening differential circular RNA expression profiles reveals the regulatory role of circTCF25-miR-103a-3p/miR-107-CDK6 pathway in bladder carcinoma. Sci Rep 6(November 2015):1–12Google Scholar
  12. 12.
    Zhang ZC, Guo XL, Li X (2018) The novel roles of circular RNAs in metabolic organs. Genes Dis 5(1):16–23CrossRefGoogle Scholar
  13. 13.
    Yang P, Qiu Z, Jiang Y et al (2016) Silencing of cZNF292 circular RNA suppresses human glioma tube formation via the Wnt/β-catenin signaling pathway. Oncotarget 7(39):63449–63455PubMedPubMedCentralGoogle Scholar
  14. 14.
    Wan L, Zhang L, Fan K et al (2016) Circular RNA-ITCH suppresses lung cancer proliferation via inhibiting the Wnt/β-Catenin pathway. Biomed Res Int 2016:1579490PubMedPubMedCentralGoogle Scholar
  15. 15.
    Geng H-H, Li R, Su Y-M et al (2016) The circular RNA Cdr1as promotes myocardial infarction by mediating the regulation of miR-7a on its target genes expression. PLoS One 11(3):e0151753CrossRefGoogle Scholar
  16. 16.
    Li F, Zhang L, Li W et al (2015) Circular RNA ITCH has inhibitory effect on ESCC by suppressing the Wnt/β-catenin pathway. Oncotarget 6(8):6001–6013PubMedPubMedCentralGoogle Scholar
  17. 17.
    Kristensen LS, Hansen TB, Venø MT et al (2018) Circular RNAs in cancer: opportunities and challenges in the field. Oncogene 37(5):555–565CrossRefGoogle Scholar
  18. 18.
    Nan A, Chen L, Zhang N et al (2017) A novel regulatory network among LncRpa, CircRar1, MiR-671 and apoptotic genes promotes lead-induced neuronal cell apoptosis. Arch Toxicol 91(4):1671–1684CrossRefGoogle Scholar
  19. 19.
    Li Y, Zheng F, Xiao X, et al (2017) CircHIPK3 sponges miR-558 to suppress heparanase expression in bladder cancer cells. EMBO Rep:e201643581-e201643581.Google Scholar
  20. 20.
    Zheng Q, Bao C, Guo W et al (2016) Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nat Commun 7:1–13Google Scholar
  21. 21.
    Chen J (2016) Circular RNA profile identifies circPVT1 as a proliferative factor and prognostic marker in gastric cancer. (December):2016–2016Google Scholar
  22. 22.
    Hansen TB, Jensen TI, Clausen BH et al (2013) Natural RNA circles function as efficient microRNA sponges. Nature 495:384CrossRefGoogle Scholar
  23. 23.
    Wang Y, Liu J, Liu C et al (2013) MicroRNA-7 regulates the mTOR pathway and proliferation in adult pancreatic β-cells. Diabetes 62(3):887–895CrossRefGoogle Scholar
  24. 24.
    Salzman J, Chen RE, Olsen MN et al (2013) Cell-type specific features of circular RNA expression. PLoS Genet 9(9):e1003777CrossRefGoogle Scholar
  25. 25.
    Darbani B, Noeparvar S, Borg S (2016) Identification of circular RNAs from the parental genes involved in multiple aspects of cellular metabolism in barley. Front Plant Sci 7(June):1–8Google Scholar
  26. 26.
    Ye CY, Chen L, Liu C et al (2015) Widespread noncoding circular RNAs in plants. New Phytol 208(1):88–95CrossRefGoogle Scholar
  27. 27.
    Sun X, Wang L, Ding J et al (2016) Integrative analysis of Arabidopsis thaliana transcriptomics reveals intuitive splicing mechanism for circular RNA. FEBS Lett 590(20):3510–3516CrossRefGoogle Scholar
  28. 28.
    Han Y-N, Xia S-Q, Zhang Y-Y et al (2017) Circular RNAs: a novel type of biomarker and genetic tools in cancer. Oncotarget 8(38):64551–64563PubMedPubMedCentralGoogle Scholar
  29. 29.
    Yang W, Du WW, Li X et al (2016) Foxo3 activity promoted by non-coding effects of circular RNA and Foxo3 pseudogene in the inhibition of tumor growth and angiogenesis. Oncogene 35(30):3919–3931CrossRefGoogle Scholar
  30. 30.
    Ashwal-Fluss R, Meyer M, Pamudurti NR et al (2014) CircRNA biogenesis competes with pre-mRNA splicing. Mol Cell 56(1):55–66PubMedPubMedCentralGoogle Scholar
  31. 31.
    Kramer MC, Liang D, Tatomer DC et al (2015) Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins. Genes Dev 29(20):2168–2182CrossRefGoogle Scholar
  32. 32.
    Du WW, Yang W, Chen Y et al (2017) Foxo3 circular RNApromotes cardiac senescence by modulating multiple factors associated with stress and senescence responses. Eur Heart J 38(18):1402–1412Google Scholar
  33. 33.
    Du WW, Yang W, Liu E et al (2016) Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2. Nucleic Acids Res 44(6):2846–2858CrossRefGoogle Scholar
  34. 34.
    Braunschweig U, Barbosa-Morais NL, Pan Q et al (2014) Widespread intron retention in mammals functionally tunes transcriptomes. Genome Res 24(11):1774–1786CrossRefGoogle Scholar
  35. 35.
    Yang Y, Fan X, Mao M et al (2017) Extensive translation of circular RNAs driven by N 6 -methyladenosine. Cell Res 27(5):626–641CrossRefGoogle Scholar
  36. 36.
    Cui X, Niu W, Kong L et al (2016) hsa_circRNA_103636: potential novel diagnostic and therapeutic biomarker in major depressive disorder. Biomark Med 10(9):943–952CrossRefGoogle Scholar
  37. 37.
    Guo X-Y, Chen J-N, Sun F et al (2017) circRNA_0046367 prevents Hepatoxicity of lipid peroxidation: an inhibitory role against hepatic steatosis. Oxid Med Cell Longev 2017:3960197CrossRefGoogle Scholar
  38. 38.
    Hansen TB, Kjems J, Damgaard CK (2013) Circular RNA and miR-7 in Cancer. Cancer Res 73(18):5609–5612CrossRefGoogle Scholar
  39. 39.
    Tang C-M, Zhang M, Huang L et al (2017) CircRNA_000203 enhances the expression of fibrosis-associated genes by derepressing targets of miR-26b-5p, Col1a2 and CTGF, in cardiac fibroblasts. Sci Rep 7:40342CrossRefGoogle Scholar
  40. 40.
    Hansen TB, Venø MT, Damgaard CK et al (2016) Comparison of circular RNA prediction tools. Nucleic Acids Res 44(6):e58–e58CrossRefGoogle Scholar
  41. 41.
    Szabo L, Salzman J (2016) Detecting circular RNAs: Bioinformatic and experimental challenges. Nat Rev Genet 17:679–692CrossRefGoogle Scholar
  42. 42.
    Quail MA, Kozarewa I, Smith F et al (2008) A large genome centre’s improvements to the Illumina sequencing system. Nat Methods 5(12):1005–1010CrossRefGoogle Scholar
  43. 43.
    Houseley J, Tollervey D (2010) Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro. PLoS One 5(8):e12271CrossRefGoogle Scholar
  44. 44.
    Luo GX, Taylor J (1990) Template switching by reverse transcriptase during DNA synthesis. J Virol 64(9):4321–4328PubMedPubMedCentralGoogle Scholar
  45. 45.
    Kelleher CD, Champoux JJ (1998) Characterization of RNA strand displacement synthesis by Moloney murine leukemia virus reverse transcriptase. J Biol Chem 273(16):9976–9986CrossRefGoogle Scholar
  46. 46.
    Panda AC, De S, Grammatikakis I et al (2017) High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs. Nucleic Acids Res 45(12):e116–e116CrossRefGoogle Scholar
  47. 47.
    Barrett SP, Salzman J (2016) Circular RNAs: analysis, expression and potential functions. Development 143(11):1838CrossRefGoogle Scholar
  48. 48.
    Zheng Y, Zhao F (2018) Detection and reconstruction of circular RNAs from transcriptomic data. In: Dieterich C, Papantonis A (eds) Circular RNAs: methods and protocols. Springer, New York, pp 1–8. Scholar
  49. 49.
    Jakobi T, Dieterich C (2018) Deep computational circular RNA analytics from RNA-seq data. In: Dieterich C, Papantonis A (eds) Circular RNAs: methods and protocols. Springer, New York, pp 9–25. Scholar
  50. 50.
    Bachmayr-Heyda A, Reiner AT, Auer K et al (2015) Correlation of circular RNA abundance with proliferation – exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues. Sci Rep 5:8057CrossRefGoogle Scholar
  51. 51.
    Memczak S, Jens M, Elefsinioti A et al (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495:333CrossRefGoogle Scholar
  52. 52.
    Zhang Y, Zhang X-O, Chen T et al (2013) Circular intronic long noncoding RNAs. Mol Cell 51(6):792–806CrossRefGoogle Scholar
  53. 53.
    Zhang X-O, Wang H-B, Zhang Y et al (2014) Complementary sequence-mediated exon circularization. Cell 159(1):134–147CrossRefGoogle Scholar
  54. 54.
    Guo JU, Agarwal V, Guo H et al (2014) Expanded identification and characterization of mammalian circular RNAs. Genome Biol 15(7):409CrossRefGoogle Scholar
  55. 55.
    Jeck WR, Sorrentino JA, Wang K et al (2013) Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19(2):141–157CrossRefGoogle Scholar
  56. 56.
    Cooper DA, Cortés-López M, Miura P (2018) Genome-wide circRNA profiling from RNA-seq data. In: Dieterich C, Papantonis A (eds) Circular RNAs: methods and protocols. Springer, New York, pp 27–41. Scholar
  57. 57.
    Zhang Y, Liang W, Zhang P et al (2017) Circular RNAs: emerging cancer biomarkers and targets. J Exp Clin Cancer Res 36(1):152CrossRefGoogle Scholar
  58. 58.
    Heumüller AW, Boeckel J-N (2018) Characterization and validation of circular RNA and their host gene mRNA expression using PCR. In: Dieterich C, Papantonis A (eds) Circular RNAs: methods and protocols. Springer, New York, pp 57–67. Scholar
  59. 59.
    Panda AC, Abdelmohsen K, Gorospe M (2017) RT-qPCR detection of senescence-associated circular RNAs. Methods Mol Biol (Clifton, N.J.) 1534:79–87CrossRefGoogle Scholar
  60. 60.
    Chen D-F, Zhang L-J, Tan K et al (2018) Application of droplet digital PCR in quantitative detection of the cell-free circulating circRNAs. Biotechnol Biotechnol Equip 32(1):116–123CrossRefGoogle Scholar
  61. 61.
    Capel B, Swain A, Nicolis S et al (1993) Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell 73(5):1019–1030CrossRefGoogle Scholar
  62. 62.
    Schneider T, Schreiner S, Preußer C et al (2018) Northern blot analysis of circular RNAs. In: Dieterich C, Papantonis A (eds) Circular RNAs: methods and protocols. Springer New York, New York, pp 119–133. Scholar
  63. 63.
    Kocks C, Boltengagen A, Piwecka M et al (2018) Single-molecule fluorescence in situ hybridization (FISH) of circular RNA CDR1as. In: Dieterich C, Papantonis A (eds) Circular RNAs: methods and protocols. Springer New York, New York, pp 77–96. Scholar
  64. 64.
    Zirkel A, Papantonis A (2018) Detecting circular RNAs by RNA fluorescence in situ hybridization. In: Dieterich C, Papantonis A (eds) Circular RNAs: methods and protocols. Springer, New York, pp 69–75. Scholar
  65. 65.
    Huang M-S, Zhu T, Li L et al (2018) LncRNAs and CircRNAs from the same gene: masterpieces of RNA splicing. Cancer Lett 415:49–57CrossRefGoogle Scholar
  66. 66.
    Song X, Zhang N, Han P et al (2016) Circular RNA profile in gliomas revealed by identification tool UROBORUS. Nucleic Acids Res 44(9):e87–e87CrossRefGoogle Scholar
  67. 67.
    Izuogu OG, Alhasan AA, Alafghani HM et al (2016) PTESFinder: a computational method to identify post-transcriptional exon shuffling (PTES) events. BMC Bioinformatics 17(1):31CrossRefGoogle Scholar
  68. 68.
    Hoffmann S, Otto C, Doose G et al (2014) A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection. Genome Biol 15(2):R34CrossRefGoogle Scholar
  69. 69.
    Wang K, Singh D, Zeng Z et al (2010) MapSplice: accurate mapping of RNA-seq reads for splice junction discovery. Nucleic Acids Res 38(18):e178–e178CrossRefGoogle Scholar
  70. 70.
    Chuang T-J, Wu C-S, Chen C-Y et al (2016) NCLscan: accurate identification of non-co-linear transcripts (fusion, trans-splicing and circular RNA) with a good balance between sensitivity and precision. Nucleic Acids Res 44(3):e29–e29CrossRefGoogle Scholar
  71. 71.
    Gao Y, Wang J, Zhao F (2015) CIRI: an efficient and unbiased algorithm for de novo circular RNA identification. Genome Biol 16(1):4CrossRefGoogle Scholar
  72. 72.
    Szabo L, Morey R, Palpant NJ et al (2015) Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development. Genome Biol 16(1):126CrossRefGoogle Scholar
  73. 73.
    Gao Y, Zhang J, Zhao F (2017) Circular RNA identification based on multiple seed matching. Brief Bioinform: bbx014–bbx014Google Scholar
  74. 74.
    Westholm JO, Miura P, Olson S et al (2014) Genomewide analysis of Drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation. Cell Rep 9(5):1966–1980CrossRefGoogle Scholar
  75. 75.
    Cheng J, Metge F, Dieterich C (2016) Specific identification and quantification of circular RNAs from sequencing data. Bioinformatics 32(7):1094–1096CrossRefGoogle Scholar
  76. 76.
    Gao Y, Zhao F (2018) Computational strategies for exploring circular RNAs. Trends Genet 34:389CrossRefGoogle Scholar
  77. 77.
    Zeng X, Lin W, Guo M et al (2017) A comprehensive overview and evaluation of circular RNA detection tools. PLoS Comput Biol 13(6):e1005420CrossRefGoogle Scholar
  78. 78.
    Langmead B, Salzberg SL (2012) Fast gapped-read alignment with bowtie 2. Nat Methods 9(4):357–359CrossRefGoogle Scholar
  79. 79.
    Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25–R25CrossRefGoogle Scholar
  80. 80.
    Li H, Durbin R (2009) Fast and accurate short read alignment with burrows–wheeler transform. Bioinformatics 25(14):1754–1760CrossRefGoogle Scholar
  81. 81.
    Dobin A, Davis CA, Schlesinger F et al (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15–21CrossRefGoogle Scholar
  82. 82.
    Hansen TB (2018) Improved circRNA identification by combining prediction algorithms. Front Cell Dev Biol 6:20CrossRefGoogle Scholar
  83. 83.
    Baruzzo G, Hayer KE, Kim EJ et al (2017) Simulation-based comprehensive benchmarking of RNA-seq aligners. Nat Methods 14(2):135–139CrossRefGoogle Scholar
  84. 84.
    Kim D, Pertea G, Trapnell C et al (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14(4):R36CrossRefGoogle Scholar
  85. 85.
    Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25(9):1105–1111CrossRefGoogle Scholar
  86. 86.
    Glažar P, Papavasileiou P, Rajewsky N (2014) circBase: a database for circular RNAs. RNA 20(11):1666–1670CrossRefGoogle Scholar
  87. 87.
    Ghosal S, Das S, Sen R et al (2013) Circ2Traits: a comprehensive database for circular RNA potentially associated with disease and traits. Front Genet 4:283CrossRefGoogle Scholar
  88. 88.
    Liu Y-C, Li J-R, Sun C-H et al (2016) CircNet: a database of circular RNAs derived from transcriptome sequencing data. Nucleic Acids Res 44(Database issue):D209–D215CrossRefGoogle Scholar
  89. 89.
    Gao Y, Wang J, Zheng Y et al (2016) Comprehensive identification of internal structure and alternative splicing events in circular RNAs. Nat Commun 7:12060CrossRefGoogle Scholar
  90. 90.
    Metge F, Czaja-Hasse LF, Reinhardt R et al (2017) FUCHS—towards full circular RNA characterization using RNAseq. PeerJ 5:e2934CrossRefGoogle Scholar
  91. 91.
    Meng X, Chen Q, Zhang P et al (2017) CircPro: an integrated tool for the identification of circRNAs with protein-coding potential. Bioinformatics 33(20):3314–3316CrossRefGoogle Scholar
  92. 92.
    Feng J, Xiang Y, Xia S, et al (2017) CircView: a visualization and exploration tool for circular RNAs. Brief Bioinform:bbx070-bbx070Google Scholar
  93. 93.
    Dudekula DB, Panda AC, Grammatikakis I et al (2016) CircInteractome: a web tool for exploring circular RNAs and their interacting proteins and microRNAs. RNA Biol 13(1):34–42CrossRefGoogle Scholar
  94. 94.
    Grant GR, Farkas MH, Pizarro AD et al (2011) Comparative analysis of RNA-Seq alignment algorithms and the RNA-Seq unified mapper (RUM). Bioinformatics 27(18):2518–2528CrossRefGoogle Scholar
  95. 95.
    Streit S, Michalski CW, Erkan M et al (2008) Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues. Nat Protoc 4:37CrossRefGoogle Scholar
  96. 96.
    PCvdC M, Roeland WD, PMvG R et al (1998) Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification. J Histochem Cytochem 46(11):1249–1259CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Siti Aishah Sulaiman
    • 1
  • Nor Azian Abdul Murad
    • 1
    Email author
  • Ezanee Azlina Mohamad Hanif
    • 1
  • Nadiah Abu
    • 1
    Email author
  • Rahman Jamal
    • 1
  1. 1.Universiti Kebangsaan Malaysia (UKM) Medical Molecular Biology Institute (UMBI)Kuala LumpurMalaysia

Personalised recommendations