Expression profiles of circular RNAs in sheep skeletal muscle

  • Cao, Yang (College of Life Sciences, Shihezi University) ;
  • You, Shuang (College of Life Sciences, Shihezi University) ;
  • Yao, Yang (College of Life Sciences, Shihezi University) ;
  • Liu, Zhi-Jin (College of Life Sciences, Shihezi University) ;
  • Hazi, Wureli (College of Animal Science and Technology, Shihezi University) ;
  • Li, Cun-Yuan (College of Life Sciences, Shihezi University) ;
  • Zhang, Xiang-Yu (College of Life Sciences, Shihezi University) ;
  • Hou, Xiao-Xu (College of Life Sciences, Shihezi University) ;
  • Wei, Jun-Chang (College of Life Sciences, Shihezi University) ;
  • Li, Xiao-Yue (College of Life Sciences, Shihezi University) ;
  • Wang, Da-Wei (College of Life Sciences, Shihezi University) ;
  • Chen, Chuang-Fu (College of Animal Science and Technology, Shihezi University) ;
  • Zhang, Yun-Feng (College of Animal Science and Technology, Shihezi University) ;
  • Ni, Wei (College of Life Sciences, Shihezi University) ;
  • Hu, Sheng-Wei (College of Life Sciences, Shihezi University)
  • Received : 2017.07.29
  • Accepted : 2018.03.13
  • Published : 2018.10.01


Objective: Circular RNAs (circRNAs) are a newfound class of non-coding RNA in animals and plants. Recent studies have revealed that circRNAs play important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports about muscle development-related circRNAs in livestock. Methods: RNA sequencing analysis was employed to identify and annotate circRNAs from longissimus dorsi of sheep. Reverse transcription followed by real-time quantitative (q) polymerase chain reaction (PCR) analysis verified the presence of these circRNAs. Targetscan7.0 and miRanda were used to analyse the interaction of circRNA-microRNA (miRNA). To investigate the function of circRNAs, an experiment was conducted to perform enrichment analysis hosting genes of circRNAs using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. Results: About 75.5 million sequences were obtained from RNA libraries of sheep skeletal muscle. These sequences were mapped to 729 genes in the sheep reference genome. We identified 886 circRNAs, including numerous circular intronic RNAs and exonic circRNAs. Reverse transcription PCR (RT-PCR) and DNA sequencing analysis confirmed the presence of several circRNAs. Real-Time RT-PCR analysis exhibited resistance of sheep circRNAs to RNase R digestion. We found that many circRNAs interacted with muscle-specific miRNAs involved in growth and development of muscle, especially circ776. The GO and KEGG enrichment analysis showed that hosting genes of circRNAs was involved in muscle cell development and signaling pathway. Conclusion: The study provides comprehensive expression profiles of circRNAs in sheep skeletal muscle. Our study offers a large number of circRNAs to facilitate a better understanding of their roles in muscle growth. Meanwhile, we suggested that circ776 could be analyzed in future study.


Supported by : National Natural Science Foundation of China (NSFC)


  1. Jeck WR, Sharpless NE. Detecting and characterizing circular RNAs. Nat Biotechnol 2014;32:453-61.
  2. Salzman J1, Gawad C, Wang PL, Lacayo N, Brown PO. Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One 2012;7:e30733.
  3. Hsu MT, Cocaprados M. Electron microscopic evidence for the circular form of RNA in the cytoplasm of eukaryotic cells. Nature 1979;280(5720):339-40.
  4. Li Z, Huang C, Bao C, et al. Exon-intron circular RNAs regulate transcription in the nucleus. Nat Struct Mol Biol 2015;22:256-64.
  5. Hansen TB, Jensen TI, Clausen BH, et al. Natural RNA circles function as efficient microRNA sponges. Nature 2013;495(7441):384-8.
  6. Rybak-Wolf A, Stottmeister C, Glazar P, et al. Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed. Mol Cell 2015;58:870-85.
  7. Fan X, Zhang X, Wu X, et al. Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos. Genome Biol 2015;16:148.
  8. Broadbent KM, Broadbent JC, Ribacke U, et al. Strand-specific RNA sequencing in Plasmodium falciparum malaria identifies developmentally regulated long non-coding RNA and circular RNA. BMC Genomics 2015;16:454.
  9. Danan M, Schwartz S, Edelheit S, Sorek R. Transcriptomewide discovery of circular RNAs in Archaea. Nucleic Acids Res 2012;40:3131-42.
  10. Lu T, Cui L, Zhou Y, et al. Transcriptome-wide investigation of circular RNAs in rice. RNA 2015;21:2076-87.
  11. Abdelmohsen K, Panda AC, De S, et al. CircRNAs in monkey muscle: age-dependent changes. Aging (Albany NY) 2015;7:903-10.
  12. Horak M, Novak J, Bienertova-Vasku J. Muscle-specific microRNAs in skeletal muscle development. Dev Biol 2016;410:1-13.
  13. Cesana M, Cacchiarelli D, Legnini I, et al. A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA. Cell 2011;147:358-69.
  14. Kim D, Pertea G, Trapnell C, et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 2013;14:R36.
  15. Zhang XO, Wang HB, Zhang Y, et al. Complementary sequencemediated exon circularization. Cell 2014;159:134-47.
  16. Agarwal V, Bell GW, Nam J, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. Elife 2015;4:e05005.
  17. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009;4:44-57.
  18. Xie C, Mao X, Huang J, et al. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res 2011; 39(Web Server issue):W316-22.
  19. Hu S, Ni W, Sai W, et al. Knockdown of myostatin expression by RNAi enhances muscle growth in transgenic sheep. PLoS One 2013;8:e58521.
  20. Chen W, Schuman E. Circular RNAs in brain and other tissues: a functional enigma. Trends Neurosci 2016;39:597-604.
  21. Zheng Q, Bao C, Guo W, et al. Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nat Commun 2016;7:11215.
  22. Bonizzato A, Gaffo E, Te Kronnie G, Bortoluzzi S. CircRNAs in hematopoiesis and hematological malignancies. Blood Cancer J 2016;6:e483.
  23. Liang Guoming, Yang Yalan, Niu Guanglin, et al. Genome-wide profiling of Sus scrofa circular RNAs acrossnine organs and three developmental stages. DNA Res 2017;24:523-35.
  24. Sun J, Xie M, Huang Z, et al. Integrated analysis of non-coding RNA and mRNA expression profiles of 2 pig breeds differing in muscle traits. J Anim Sci 2017;95:1092-103.
  25. Estrella NL, Desjardins CA, Nocco SE, et al. MEF2 transcription factors regulate distinct gene programs in mammalian skeletal muscle differentiation. J Biol Chem 2015;290:1256-68.
  26. Liu N, Nelson BR, Bezprozvannaya S, et al. Requirement of MEF2A, C, and D for skeletal muscle regeneration. Proc Natl Acad Sci USA 2014;111:4109-14.
  27. Ornatsky OI, Andreucci JJ, McDermott JC. A dominant-negative form of transcription factor MEF2 inhibits myogenesis. J Biol Chem 1997;272:33271-8.
  28. Seok HY, Tatsuguchi M, Callis TE, et al. miR-155 inhibits expression of the MEF2A protein to repress skeletal muscle differentiation. J Biol Chem 2011;286:35339-46.
  29. Salih DA, Tripathi G, Holding C, et al. Insulin-like growth factor-binding protein 5 (IGFBP5) compromises survival, growth, muscle development, and fertility in mice. Proc Natl Acad Sci USA 2004;101:4314-9.
  30. Awede B, Thissen JP, Gailly P, Lebacq, J. Regulation of IGF-I, IGFBP-4 and IGFBP-5 gene expression by loading in mouse skeletal muscle. FEBS Lett 1999;461:263-7.
  31. Zhang WR, Zhang HN, Wang YM, et al. miR-143 regulates proliferation and differentiation of bovine skeletal muscle satellite cells by targeting IGFBP5. In Vitro Cell Dev Biol Anim 2017;53:265-71.
  32. Soriano-Arroquia A, McCormick R, Molloy AP, et al. Agerelated changes in miR-143-3p:Igfbp5 interactions affect muscle regeneration. Aging Cell 2016;15:361-9.
  33. Cristiano BE, Chan JC, Hannan KM, et al. A specific role for AKT3 in the genesis of ovarian cancer through modulation of G2-M phase transition. Cancer Res 2006;66:11718-25.
  34. Matheny Ronald W, Adamo Martin L. Role of Akt isoforms in IGF-I-mediated signaling and survival in myoblasts. Biochem Biophys Res Commun 2009;389:117-21.
  35. Micke GC, Sullivan TM, Mcmillen IC, Gentili S, Perry VE. Protein intake during gestation affects postnatal bovine skeletal muscle growth and relative expression of IGF1, IGF1R, IGF2 and IGF2R. Mol Cell Endocrinol 2011;332:234-41.
  36. Deng YY, Zhang W, She J, et al. GW27-e1167 Circular RNA related to PPAR${\gamma}$ function as ceRNA of microRNA in human acute myocardial infarction. J Am Coll Cardiol 2016;68:C51-2.
  37. Kartha RV, Subramanian S. Competing endogenous RNAs (ceRNAs): new entrants to the intricacies of gene regulation. Front Genet 2014;5:8.
  38. Wei W, He HB, Zhang WY, et al. miR-29 targets Akt3 to reduce proliferation and facilitate differentation of myoblasts in skeletal muscle development. Cell Death Dis 2013;4:e668.
  39. Wong CF, Tellam RL. MicroRNA-26a targets the histone methyltransferase Enhancer of Zeste homolog 2 during myogenesis. J Biol Chem 2008;283:9836-43.
  40. Wang B, Zhang C, Zhang A, et al. MicroRNA-23a and MicroRNA-27a mimic exercise by ameliorating CKD-induced muscle atrophy. J Am Soc Nephrol 2017;28:2631-40.
  41. Liu B, Shi Y, He H, et al. miR-221 modulates skeletal muscle satellite cells proliferation and differentiation. In Vitro Cell Dev Biol Anim 2018;54:147-55.
  42. Chen JF, Mandel EM, Thomson JM, et al. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet 2006;38:228-33.
  43. Feng Y, Niu L-L, Wei W, et al. A feedback circuit between miR-133 and the ERK1/2 pathway involving an exquisite mechanism for regulating myoblast proliferation and differentiation. Cell Death Dis 2013;4:e934.
  44. Legnini I, Di Timoteo G, Rossi F, et al. Circ-ZNF609 is a circular RNA that can be translated and functions in myogenesis. Mol Cell 2017;66:22-37.e9.
  45. Wei X, Li H, Yang J, et al. Circular RNA profiling reveals an abundant circLMO7 that regulates myoblasts differentiation and survival by sponging miR-378a-3p. Cell Death Dis 2017;8:e3153.
  46. Werfel S, Nothjunge S, Schwarzmayr T, et al. Characterization of circular RNAs in human, mouse and rat hearts. J Mol Cell Cardiol 2016;98:103-7.

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