Skip to main content
SpringerLink
Log in

The Guideline of the Design and Validation of MiRNA Mimics

  • Protocol
  • First Online:
MicroRNA and Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 676))

Abstract

The miRNA mimic technology (miR-Mimic) is an innovative approach for gene silencing. This approach is to generate nonnatural double-stranded miRNA-like RNA fragments. Such an RNA fragment is designed to have its 5′-end bearing a partially complementary motif to the selected sequence in the 3′UTR unique to the target gene. Once introduced into cells, this RNA fragment, mimicking an endogenous miRNA, can bind specifically to its target gene and produce posttranscriptional repression, more specifically translational inhibition, of the gene. Unlike endogenous miRNAs, miR-Mimics act in a gene-specific fashion. The miR-Mimic approach belongs to the “miRNA-targeting” and “miRNA-gain-of-function” strategy and is primarily used as an exogenous tool to study gene function by targeting mRNA through miRNA-like actions in mammalian cells. The technology was developed by my research group (Department of Medicine, Montreal Heart Institute, University of Montreal) in 2007 (Xiao, et al. J Cell Physiol 212:285–292, 2007; Xiao et al. Nat Cell Biol, in review).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Xiao J, Yang B, Lin H, Lu Y, Luo X, Wang Z (2007) Novel approaches for gene-specific interference via manipulating actions of microRNAs: examination on the pacemaker channel genes HCN2 and HCN4. J Cell Physiol 212:285–292.

    Article  PubMed  CAS  Google Scholar 

  2. Xiao J, Lin H, Luo X, Chen G, Wang Z (2009) microRNA-605 joins p53:Mdm2 network to form a positive feedback loop in cell-fate decision. EMBO J (accepted).

    Google Scholar 

  3. Xia H, Mao Q, Paulson HL, Davidson BL (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 20:1006–1010.

    Article  PubMed  CAS  Google Scholar 

  4. Golden DE, Gerbasi VR, Sontheimer EJ (2008) An inside job for siRNAs. Mol Cell 31:309–312.

    Article  PubMed  CAS  Google Scholar 

  5. Pushparaj PN, Aarthi JJ, Manikandan J, Kumar SD (2008) siRNA, miRNA, and shRNA: in vivo applications. J Dent Res 87:992–1003.

    Article  PubMed  CAS  Google Scholar 

  6. Wang Z, Luo X, Lu Y, Yang B (2008) miRNAs at the heart of the matter. J Mol Med 86:771–783.

    Article  PubMed  CAS  Google Scholar 

  7. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB (2003) Prediction of mammalian microRNA targets. Cell 115:787–798.

    Article  PubMed  CAS  Google Scholar 

  8. Doench JG, Sharp PA (2004) Specificity of microRNA target selection in translational repression. Genes Dev 18:504–511.

    Article  PubMed  CAS  Google Scholar 

  9. Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G, Mello CC (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106:23–34.

    Article  PubMed  CAS  Google Scholar 

  10. Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A, Mello CC (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99:123–132.

    Article  PubMed  CAS  Google Scholar 

  11. Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17:438–442.

    Article  PubMed  CAS  Google Scholar 

  12. Hutvágner G, Zamore PD (2002) A microRNA in a multiple-turnover RNAi enzyme complex. Science 297:2056–2060.

    Article  PubMed  Google Scholar 

  13. Zeng Y, Yi R, Cullen BR (2003) MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci USA 100:9779–9784.

    Article  PubMed  CAS  Google Scholar 

  14. Brennecke J, Stark A, Russell RB, Cohen SM (2005) Principles of microRNA–target recognition. PLoS Biol 3:404–418.

    Article  CAS  Google Scholar 

  15. Kiriakidou M, Tan GS, Lamprinaki S, De Planell-Saguer M, Nelson PT, Mourelatos Z (2007) An mRNA m(7)G cap binding-like motif within human Ago2 represses translation. Cell 129:1141–1151.

    Article  PubMed  CAS  Google Scholar 

  16. Kloosterman WP, Wienholds E, Ketting RF, Plasterk RH (2004) Substrate requirements for let-7 function in the developing zebrafish embryo. Nucleic Acids Res 32:6284–6291.

    Article  PubMed  CAS  Google Scholar 

  17. Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200.

    Article  PubMed  CAS  Google Scholar 

  18. Haley B, Zamore PD (2004) Kinetic analysis of the RNAi enzyme complex. Nat Struct Mol Biol 11:599–606.

    Article  PubMed  CAS  Google Scholar 

  19. Martinez J, Tuschl T (2004) RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes Dev 18:975–980.

    Article  PubMed  CAS  Google Scholar 

  20. Kiriakidou M, Nelson PT, Kouranov A, Fitziev P, Bouyioukos C, Mourelatos Z, Hatzigeorgiou A (2004) A combined computational–experimental approach predicts human microRNA targets. Genes Dev 18:1165–1178.

    Article  PubMed  CAS  Google Scholar 

  21. Ha I, Wightman B, Ruvkun G (1996) A bulged lin-4/lin-14 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation. Genes Dev 10:3041–3050.

    Article  PubMed  CAS  Google Scholar 

  22. Chiu YL, Rana TM (2003) siRNA function in RNAi: a chemical modification analysis. RNA 9:1034–1048.

    Article  PubMed  CAS  Google Scholar 

  23. Czauderna F, Fechtner M, Dames S, Aygun H, Klippel A, Pronk GJ, Giese K, Kaufmann J (2003) Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res 31:2705–2716.

    Article  PubMed  CAS  Google Scholar 

  24. Prakash TP, Allerson CR, Dande P, Vickers TA, Sioufi N, Jarres R, Baker BF, Swayze EE, Griffey RH, Bhat B (2005) Positional effect of chemical modifications on short interference RNA activity in mammalian cells. J Med Chem 48:4247–4253.

    Article  PubMed  CAS  Google Scholar 

  25. Kraynack BA, Baker BF (2006) Small interfering RNAs containing full 2′-Omethylribonucle-otide-modified sense strands display Argonaute2/eIF2C2-dependent activity. RNA 12:163–176.

    Article  PubMed  CAS  Google Scholar 

  26. Allerson CR, Sioufi N, Jarres R, Prakash TP, Naik N, Berdeja A, Wanders L, Griffey RH, Swayze EE, Bhat B (2005) Fully 2′-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA. J Med Chem 48:901–904.

    Article  PubMed  CAS  Google Scholar 

  27. Layzer JM, McCaffrey AP, Tanner AK, Huang Z, Kay MA, Sullenger BA (2004) In vivo activity of nuclease-resistant siRNAs. RNA 10:766–771.

    Article  PubMed  CAS  Google Scholar 

  28. Harborth J, Elbashir SM, Vandenburgh K, Manninga H, Scaringe SA, Weber K, Tuschl T (2003) Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev 13:83–105.

    Article  PubMed  CAS  Google Scholar 

  29. Braasch DA, Jensen S, Liu Y, Kaur K, Arar K, White MA, Corey DR (2003) RNA interference in mammalian cells by chemically-modified RNA. Biochemistry 42:7967–7975.

    Article  PubMed  CAS  Google Scholar 

  30. Morrissey DV, Blanchard K, Shaw L, Jensen K, Lockridge JA, Dickinson B, McSwiggen JA, Vargeese C, Bowman K, Shaffer CS, Polisky BA, Zinnen S (2005) Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication. Hepatology 41:1349–1356.

    Article  PubMed  CAS  Google Scholar 

  31. Amarzguioui M, Holen T, Babaie E, Prydz H (2003) Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Res 31:589–595.

    Article  PubMed  CAS  Google Scholar 

  32. Altmann KH, Dean NM, Fabbro D, Freier SM, Geiger T, Haener R, Huesken D, Martin P, Monia BP, Muller M, Natt F, Nicklin P, Phillips J, Pieles U, Sasmor H, Moser H (1996) Second generation of antisense oligonucleotides. From nuclease resistance to biological efficacy in animals. Chimia 50:168–176.

    CAS  Google Scholar 

  33. Hoke GD, Draper K, Freier SM, Gonzalez C, Driver VB, Zounes MC, Ecker DJ (1991) Effects of phosphorothioate capping on antisense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection. Nucleic Acids Res 19:5743–5748.

    Article  PubMed  CAS  Google Scholar 

  34. Braasch DA, Paroo Z, Constantinescu A, Ren G, Oz OK, Mason RP, Corey DR (2004) Biodistribution of phosphodiester and phosphorothioate siRNA. Bioorg Med Chem Lett 14:1139–1143.

    Article  PubMed  CAS  Google Scholar 

  35. McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA (2002) RNA interference in adult mice. Nature 418:38–39.

    Article  PubMed  CAS  Google Scholar 

  36. Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN, Harborth J, Heyes JA, Jeffs LB, John M, Judge AD, Lam K, McClintock K, Nechev LV, Palmer LR, Racie T, Rohl I, Seiffert S, Shanmugam S, Sood V, Soutschek J, Toudjarska I, Wheat AJ, Yaworski E, Zedalis W, Koteliansky V, Manoharan M, Vornlocher HP, MacLachlan I (2006) RNAi-mediated gene silencing in non-human primates. Nature 441:111–114.

    Article  PubMed  CAS  Google Scholar 

  37. Morrissey DV, Lockridge J.A, Shaw L, Blanchard K, Jensen K, Breen W, Hartsough K, Machemer L, Radka S, Jadhav V, Vaish N, Zinnen S, Vargeese C, Bowman K, Shaffer CS, Jeffs LB, Judge A, MacLachlan I, Polisky B (2005) Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat Biotechnol 23:1002–1007.

    Article  PubMed  CAS  Google Scholar 

  38. Ge Q, Filip L, Bai A, Nguyen T, Eisen HN, Chen J (2004) Inhibition of influenza virus production in virus-infected mice by RNA interference. Proc Natl Acad Sci USA 101:8676–8681.

    Article  PubMed  CAS  Google Scholar 

  39. Yang B, Lin H, Xiao J, Luo X, Li B, Lu Y, Wang H, Wang Z (2007) The muscle-specific microRNA miR-1 causes cardiac arrhythmias by targeting GJA1 and KCNJ2 genes. Nat Med 13:486–491.

    Article  PubMed  CAS  Google Scholar 

  40. Xiao J, Luo X, Lin H, Xu C, Gao H, Wang H, Yang B, Wang Z (2007) MicroRNA miR-133 represses HERG K+ channel expression contributing to QT prolongation in diabetic hearts. J Biol Chem 282:12363–12367.

    Article  PubMed  CAS  Google Scholar 

  41. Luo X, Lin H, Lu Y, Li B, Xiao J, Yang B, Wang Z (2007) Transcriptional activation by stimulating protein 1 and post-transcriptional repression by muscle-specific microRNAs of IKs-encoding genes and potential implications in regional heterogeneity of their expressions. J Cell Physiol 212:358–367.

    Article  PubMed  CAS  Google Scholar 

  42. Luo X, Lin H, Pan Z, Xiao J, Zhang Y, Lu Y, Yang B, Wang Z (2008) Overexpression of Sp1 and downregulation of miR-1/miR-133 activates re-expression of pacemaker channel genes HCN2 and HCN4 in hypertrophic heart. J Biol Chem 283:20045–20052.

    Article  PubMed  CAS  Google Scholar 

  43. Xu C, Lu Y, Lin H, Xiao J, Wang H, Luo X, Li B, Yang B, Wang Z (2007) The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis via targeting HSP60/HSP70 and caspase-9 in cardiomyocytes. J Cell Sci 120:3045–3052.

    Article  PubMed  CAS  Google Scholar 

  44. Lu Y, Xiao J, Lin H, Bai Y, Luo X, Wang Z, Yang B (2009) Complex antisense inhibitors offer a superior approach for microRNA research and therapy. Nucleic Acids Res 37:e24–e33.

    Article  PubMed  Google Scholar 

  45. Xiao L, Xiao J, Luo X, Lin H, Wang Z, Nattel S (2008) Feedback remodeling of cardiac potassium current expression. A novel potential mechanism for control of repolarization reserve. Circulation 118:983–992.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by the Canadian Institute of Health Research, Heart and Stroke Foundation of Quebec, and Fonds de la Recherche de l’Institut de Cardiologie de Montreal. Dr. Z. Wang is a Changjiang Scholar Professor of the Ministry of Education of China and a Longjiang Scholar Professor of Heilongjiang, China. The authors thank XiaoFan Yang for her excellent technical supports.

Author information

Authors and Affiliations

  1. Department of Medicine, Montreal Heart Institute, University of Montreal, Montreal, QC, Canada

    Zhiguo Wang

Authors
  1. Zhiguo Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. , Department of Biological Science, University of Calgary, University Drive N.W. 2500, Calgary, T2N 1N4, Alberta, Canada

    Wei Wu

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Wang, Z. (2011). The Guideline of the Design and Validation of MiRNA Mimics. In: Wu, W. (eds) MicroRNA and Cancer. Methods in Molecular Biology, vol 676. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-863-8_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-863-8_15

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-862-1

  • Online ISBN: 978-1-60761-863-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation