Advertisement

Evaluating the MicroRNA Targeting Sites by Luciferase Reporter Gene Assay

  • Yi Jin
  • Zujian Chen
  • Xiqiang Liu
  • Xiaofeng ZhouEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 936)

Abstract

MicroRNAs are post-transcriptional regulators that control mRNA stability and the translation efficiency of their target genes. Mature microRNAs are approximately 22-nucleotide in length. They mediate post-transcriptional gene regulation by binding to the imperfect complementary sequences (a.k.a. microRNA regulatory elements, MRE) in the target mRNAs. It is estimated that more than one-third of the protein-coding genes in the human genome are regulated by microRNAs. The experimental methods to examine the interaction between the microRNA and its targeting site(s) in the mRNA are important for understanding microRNA functions. The luciferase reporter gene assay has recently been adapted to test the effect of microRNAs. In this chapter, we use a previously identified miR-138 targeting site in the 3′-untranslated region (3′-UTR) of the RhoC mRNA as an example to describe a quick method for testing the interaction of microRNA and mRNA.

Key words

MicroRNA MicroRNA targeting sequence MicroRNA regulatory element Luciferase reporter gene assay miR-138 RhoC 

Notes

Acknowledgment

This work was supported in part by NIH PHS grants (CA135992, CA139596, DE014847) and supplementary funding from UIC CCTS (UL1RR029879). Y.J. is supported by PHS T32DE018381 from NIDCR. We thank Ms. Katherine Long for her editorial assistance.

References

  1. 1.
    Bartel DP (2004) MicroRNAs: genomics, ­biogenesis, mechanism, and function. Cell 116: 281–297PubMedCrossRefGoogle Scholar
  2. 2.
    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136: 215–233PubMedCrossRefGoogle Scholar
  3. 3.
    Dai Y, Zhou X (2010) Computational methods for the identification of microRNA targets. Open Access Bioinform 2:29–39Google Scholar
  4. 4.
    Wang C, Li Q (2007) Identification of differentially expressed microRNAs during the development of Chinese murine mammary gland. J Genet Genomics 34:966–973PubMedCrossRefGoogle Scholar
  5. 5.
    Siegel G, Obernosterer G, Fiore R, Oehmen M, Bicker S, Christensen M, Khudayberdiev S, Leuschner PF, Busch CJ, Kane C, Hubel K, Dekker F, Hedberg C, Rengarajan B, Drepper C, Waldmann H, Kauppinen S, Greenberg ME, Draguhn A, Rehmsmeier M, Martinez J, Schratt GM (2009) A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis. Nat Cell Biol 11:705–716PubMedCrossRefGoogle Scholar
  6. 6.
    Morton SU, Scherz PJ, Cordes KR, Ivey KN, Stainier DY, Srivastava D (2008) microRNA-138 modulates cardiac patterning during embryonic development. Proc Natl Acad Sci USA 105:17830–17835PubMedCrossRefGoogle Scholar
  7. 7.
    Kisliouk T, Yosefi S, Meiri N (2011) MiR-138 inhibits EZH2 methyltransferase expression and methylation of histone H3 at lysine 27, and affects thermotolerance acquisition. Eur J Neurosci 33:224–235PubMedCrossRefGoogle Scholar
  8. 8.
    Mitomo S, Maesawa C, Ogasawara S, Iwaya T, Shibazaki M, Yashima-Abo A, Kotani K, Oikawa H, Sakurai E, Izutsu N, Kato K, Komatsu H, Ikeda K, Wakabayashi G, Masuda T (2008) Downregulation of miR-138 is associated with overexpression of human telomerase reverse transcriptase protein in human anaplastic thyroid carcinoma cell lines. Cancer Sci 99:280–286PubMedCrossRefGoogle Scholar
  9. 9.
    Seike M, Goto A, Okano T, Bowman ED, Schetter AJ, Horikawa I, Mathe EA, Jen J, Yang P, Sugimura H, Gemma A, Kudoh S, Croce CM, Harris CC (2009) MiR-21 is an EGFR-regulated anti-apoptotic factor in lung cancer in never-smokers. Proc Natl Acad Sci USA 106:12085–12090PubMedCrossRefGoogle Scholar
  10. 10.
    Zhao X, Yang L, Hu J, Ruan J (2010) miR-138 might reverse multidrug resistance of leukemia cells. Leuk Res 34:1078–1082PubMedCrossRefGoogle Scholar
  11. 11.
    Jiang L, Dai Y, Liu X, Wang C, Wang A, Chen Z, Heidbreder CE, Kolokythas A, Zhou X (2011) Identification and experimental validation of G protein alpha inhibiting activity polypeptide 2 (GNAI2) as a microRNA-138 target in tongue squamous cell carcinoma. Hum Genet 129:189–197PubMedCrossRefGoogle Scholar
  12. 12.
    Jiang L, Liu X, Kolokythas A, Yu J, Wang A, Heidbreder CE, Shi F, Zhou X (2010) Down-regulation of the Rho GTPase signaling ­pathway is involved in the microRNA-138 mediated inhibition of cell migration and invasion in tongue squamous cell carcinoma. Int J Cancer 127:505–512PubMedCrossRefGoogle Scholar
  13. 13.
    Wong TS, Liu XB, Chung-Wai Ho A, Po-Wing Yuen A, Wai-Man Ng R, Ignace Wei W (2008) Identification of pyruvate kinase type M2 as potential oncoprotein in squamous cell carcinoma of tongue through microRNA profiling. Int J Cancer 123:251–257PubMedCrossRefGoogle Scholar
  14. 14.
    Wong TS, Liu XB, Wong BY, Ng RW, Yuen AP, Wei WI (2008) Mature miR-184 as potential oncogenic microRNA of squamous cell carcinoma of tongue. Clin Cancer Res 14:2588–2592PubMedCrossRefGoogle Scholar
  15. 15.
    Kozaki K, Imoto I, Mogi S, Omura K, Inazawa J (2008) Exploration of tumor-suppressive microRNAs silenced by DNA hypermethylation in oral cancer. Cancer Res 68:2094–2105PubMedCrossRefGoogle Scholar
  16. 16.
    Liu X, Jiang L, Wang A, Yu J, Shi F, Zhou X (2009) MicroRNA-138 suppresses invasion and promotes apoptosis in head and neck squamous cell carcinoma cell lines. Cancer Lett 286:217–222PubMedCrossRefGoogle Scholar
  17. 17.
    Kawasaki H, Taira K (2003) Hes1 is a target of microRNA-23 during retinoic-acid-induced neuronal differentiation of NT2 cells. Nature 423:838–842PubMedCrossRefGoogle Scholar
  18. 18.
    Qin W, Shi Y, Zhao B, Yao C, Jin L, Ma J, Jin Y (2010) miR-24 regulates apoptosis by targeting the open reading frame (ORF) region of FAF1 in cancer cells. PLoS One 5:e9429PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Yi Jin
    • 1
  • Zujian Chen
    • 2
  • Xiqiang Liu
    • 1
  • Xiaofeng Zhou
    • 3
    Email author
  1. 1.Center for Molecular Biology of Oral DiseasesUniversity of Illinois at ChicagoChicagoUSA
  2. 2.Department of Anatomy and Cell BiologyRush University Medical CenterChicagoUSA
  3. 3.Center for Molecular Biology of Oral Diseases, College of DentistryUniversity of Illinois at ChicagoChicagoUSA

Personalised recommendations