Abstract
Single microRNA (miRNA) can be inhibited using antagomiR which efficiently knockdown a specific miRNA. However, the effect is transient and often results in subtle phenotype. Here we report a guideline on designing miRNA sponge inhibiting a miRNA family. As a model system, we targeted miR-30 family, known as tumor suppressor miRNAs in multiple tumors. To achieve an efficient knockdown, we generated perfect and bulged-matched miRNA binding sites (MBS) and introduced multiple copies of MBS. The protocol here demonstrates the miRNA sponge as a useful tool to examine the functional impact of inhibition miRNAs.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Eulalio A, Huntzinger E, Izaurralde E (2008) Getting to the root of miRNA-mediated gene silencing. Cell 132:9–14
Georges M, Coppieters W, Charlier C (2007) Polymorphic miRNA-mediated gene regulation: contribution to phenotypic variation and disease. Curr Opin Genet Dev 17:166–176
Rai D, Karanti S, Junk I, Dahia P, Aguiar RC (2008) Coordinated expression of microRNA-155 and predicted target genes in DLBCL. Cancer Genet Cytogenet 181:8–15
Chaubey A, Karanti S, Rai D, Oh T, Adhvaryu SG, Aguiar RC (2009) MicroRNAs and deletion of the derivative chromosome 9 in chronic myeloid leukemia. Leukemia 23:186–188
Jung I, Aguiar RC (2009) MicroRNA-155 expression and outcome in diffuse large B-cell lymphoma. Br J Haematol 144:138–140
Li C, Kim SW, Rai D, Bolla A, Kinney M, Robetorye R, Aguiar RC (2009) Copy number abnormalities, MYC activity and the fingerprint of normal B-cells define the microRNA profile of DLBCL. Blood 113:6681–6690
Rai D, Kim SW, McKeller MR, Dahia PLM, Aguiar RC (2010) Targeting of SMAD5 links microRNA-155 to the TGFβ pathway and lymphomagenesis. Proc Natl Acad Sci U S A 107:3111–3116
Kim SW, Ramasamy K, Bouamar H, Lin AP, Jiang D, Aguiar RC (2012) MicroRNAs miR-125a and miR-125b constitutively activate the NF-kB pathway by targeting the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20). Proc Natl Acad Sci USA 109:7865–7870
Jiang D, Aguiar RC (2014) MicroRNA-155 controls RB phosphorylation in normal and malignant B lymphocytes via the non-canonical TGFB1-SMAD5 signaling module. Blood 123:86–93
Ortega M, Bhatnagar H, Lin AP, Wang L, Aster JC, Sill H, Aguiar RC (2015) A microRNA-mediated regulatory loop modulates NOTCH and MYC oncogenic signals in B- and T-cell malignancies. Leukemia 29:968–976
Jeong D, Kim J, Nam J, Sun H, Lee YH, Lee TJ, Aguiar RC, Kim SW (2015) MicroRNA-124 links p53 to the NF-kB pathway in B cell lymphomas. Leukemia 29:1868–1874
Bouamar H, Jiang D, Wang L, Lin AP, Ortega M, Aguiar RC (2015) MicroRNA-155 control of p53 activity is context dependent and mediated by Aicda and Socs1. Mol Cell Biol 35:1329–1340
Ebert MS, Neilson JR, Sharp PA (2007) MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods 4:721–726
Piva R, Spandidos DA, Gambari R (2013) From microRNA functions to microRNA therapeutics: novel targets and novel drugs in breast cancer research and treatment (Review). Int J Oncol 43:985–994
Esau CC (2008) Inhibition of microRNA with antisense oligonucleotides. Methods 44:55–60
Takahashi M, Yamada N, Hatakeyama H, Murata M, Sato Y, Minakawa N, Harashima H, Matsuda A (2013) In vitro optimization of 2′-OMe-4′-thioribonucleoside-modified anti-microRNA oligonucleotides and its targeting delivery to mouse liver using a liposomal nanoparticle. Nucleic Acids Res 41:10659–10667
Lennox KA, Owczarzy R, Thomas DM, Walder JA, Behlke MA (2013) Improved performance of anti-miRNA oligonucleotides using a novel non-nucleotide modifier. Mol Ther Nucleic Acids 2:e117
Wu SQ, Xu ZZ, Lin J, Zhan R (2012) Construction of miRNA sponge targeting miR-20a and stable expression in Jurkat leukemia cell line. J Exp Hematol 20:1056–1062
Chen L, Zhang K, Shi Z, Zhang A, Jia Z, Wang G, Pu P, Kang C, Han L (2014) A lentivirus-mediated miR-23b sponge diminishes the malignant phenotype of glioma cells in vitro and in vivo. Oncol Rep 31:1573–1580
Kluiver J, Slezak-Prochazka I, Smigielska-Czepiel K, Halsema N, Kroesen BJ, van den Berg A (2012) Generation of miRNA sponge constructs. Methods 58:113–117
Simcox TG, Fabian L, Kretz K, Hedden V, Simcox ME (1995) SanDI, a new type-II restriction endonuclease that recognizes 5′-GG/GWCCC-3′. Gene 155:129–130
Jung J, Yeom C, Choi YS, Kim S, Lee E, Park MJ, Kang SW, Kim SB, Chang S (2015) Simultaneous inhibition of multiple oncogenic miRNAs by a multi-potent microRNA sponge. Oncotarget 6:20370–20387
Acknowledgement
We thank Dr. Ricardo CT Aguiar from The University of Texas Health Science Center at San Antonio, USA, for the valuable advices during the sponge constructions.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Ortega, M.M., Bouamar, H. (2017). Guidelines on Designing MicroRNA Sponges: From Construction to Stable Cell Line. In: Rani, S. (eds) MicroRNA Profiling. Methods in Molecular Biology, vol 1509. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6524-3_20
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6524-3_20
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6522-9
Online ISBN: 978-1-4939-6524-3
eBook Packages: Springer Protocols