MicroRNA (miRNA) sponges are RNA transcripts containing multiple high-affinity binding sites that associate with and sequester specific miRNAs to prevent them from interacting with their target messenger (m)RNAs. Due to the high specificity of miRNA sponges and strong inhibition of target miRNAs, these molecules have become increasingly applied in miRNA loss-of-function studies. However, improperly designed sponge constructs may sequester off-target miRNAs; thus, it has become increasingly important to develop a tool for miRNA sponge construct design and testing. In this study, we introduce microRNA sponge generator and tester (miRNAsong), a freely available web-based tool for generation and in silico testing of miRNA sponges. This tool generates miRNA sponge constructs for specific miRNAs and miRNA families/clusters and tests them for potential binding to miRNAs in selected organisms. Currently, miRNAsong allows for testing of sponge constructs in 219 species covering 35,828 miRNA sequences. Furthermore, we also provide an example, supplemented with experimental data, of how to use this tool. Using miRNAsong, we designed and tested a sponge for miR-145 inhibition, and cloned the sequence into an inducible lentiviral vector. We found that established cell lines expressing miR-145 sponge strongly inhibited miR-145, thus demonstrating the usability of miRNAsong tool for sponge generation. URL: http://www.med.muni.cz/histology/miRNAsong/.

Department of Histology and Embryology Faculty of Medicine Masaryk University Kamenice 3 625 00 Brno Czech Republic

International Clinical Research Center St Anne's University Hospital Brno Pekařská 53 656 91 Brno Czech Republic

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Barta T. et al.. Brief Report: Inhibition of miR-145 Enhances Reprogramming of Human Dermal Fibroblasts to Induced Pluripotent Stem Cells. Stem Cells Dayt. Ohio 34, 246–251 (2016). PubMed PMC

Dolezalova D. et al.. MicroRNAs regulate p21(Waf1/Cip1) protein expression and the DNA damage response in human embryonic stem cells. Stem Cells Dayt. Ohio 30, 1362–1372 (2012). PubMed

Wilfred B. R., Wang W.-X. & Nelson P. T. Energizing miRNA research: a review of the role of miRNAs in lipid metabolism, with a prediction that miR-103/107 regulates human metabolic pathways. Mol. Genet. Metab. 91, 209–217 (2007). PubMed PMC

Li C., Feng Y., Coukos G. & Zhang L. Therapeutic microRNA strategies in human cancer. AAPS J. 11, 747–757 (2009). PubMed PMC

Musilova K. & Mraz M. MicroRNAs in B-cell lymphomas: how a complex biology gets more complex. Leukemia 29, 1004–1017 (2015). PubMed

Dyawanapelly S., Ghodke S. B., Vishwanathan R., Dandekar P. & Jain R. RNA interference-based therapeutics: molecular platforms for infectious diseases. J. Biomed. Nanotechnol. 10, 1998–2037 (2014). PubMed

Kaboli P. J., Rahmat A., Ismail P. & Ling K.-H. MicroRNA-based therapy and breast cancer: A comprehensive review of novel therapeutic strategies from diagnosis to treatment. Pharmacol. Res. 97, 104–121 (2015). PubMed

Luck M. E., Muljo S. A. & Collins C. B. Prospects for Therapeutic Targeting of MicroRNAs in Human Immunological Diseases. J. Immunol. Baltim. Md 1950 194, 5047–5052 (2015). PubMed PMC

Monroig-Bosque P. del C., Rivera C. A. & Calin G. A. MicroRNAs in cancer therapeutics: ‘from the bench to the bedside’. Expert Opin. Biol. Ther. 15, 1381–1385 (2015). PubMed PMC

Ebert M. S. & Sharp P. A. MicroRNA sponges: Progress and possibilities. RNA 16, 2043–2050 (2010). PubMed PMC

Mukherji S. et al.. MicroRNAs can generate thresholds in target gene expression. Nat. Genet. 43, 854–859 (2011). PubMed PMC

Kluiver J. et al.. Generation of miRNA sponge constructs. Methods San Diego Calif 58, 113–117 (2012). PubMed

Ebert M. S., Neilson J. R. & Sharp P. A. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat. Methods 4, 721–726 (2007). PubMed PMC

Esau C. C. Inhibition of microRNA with antisense oligonucleotides. Methods San Diego Calif 44, 55–60 (2008). PubMed

Kluiver J. et al.. Rapid generation of microRNA sponges for microRNA inhibition. PloS One 7, e29275 (2012). PubMed PMC

Gentner B. et al.. Stable knockdown of microRNA in vivo by lentiviral vectors. Nat. Methods 6, 63–66 (2009). PubMed

Haraguchi T., Ozaki Y. & Iba H. Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells. Nucleic Acids Res. 37, e43 (2009). PubMed PMC

Athyros V. G., Katsiki N. & Karagiannis A. Is Targeting microRNAs the Philosopher’s Stone for Vascular Disease ?  Curr. Vasc. Pharmacol. 14, 88–97 (2016). PubMed

Wang X.-W. et al.. MicroRNA-221 sponge therapy attenuates neointimal hyperplasia and improves blood flows in vein grafts. Int. J. Cardiol. 208, 79–86 (2016). PubMed

Zhuang C.-L. et al.. Synthetic miRNA sponges driven by mutant hTERT promoter selectively inhibit the progression of bladder cancer. Tumour Biol. J. Int. Soc. Oncodevelopmental Biol. Med. 36, 5157–5163 (2015). PubMed

Londin E. et al.. Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- and tissue-specific microRNAs. Proc. Natl. Acad. Sci. USA 112, E1106–E1115 (2015). PubMed PMC

Gaillet B. et al.. High-level recombinant protein production in CHO cells using lentiviral vectors and the cumate gene-switch. Biotechnol. Bioeng. 106, 203–215 (2010). PubMed

Mullick A. et al.. The cumate gene-switch: a system for regulated expression in mammalian cells. BMC Biotechnol. 6, 43 (2006). PubMed PMC

Griffiths-Jones S., Saini H. K., Dongen S. van & Enright A. J. miRBase: tools for microRNA genomics. Nucleic Acids Res. 36, D154–D158 (2008). PubMed PMC

Griffiths-Jones S., Grocock R. J., Dongen S. van, Bateman A. & Enright A. J. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 34, D140–D144 (2006). PubMed PMC

Griffiths‐Jones S. The microRNA Registry. Nucleic Acids Res. 32, D109–D111 (2004). PubMed PMC

Kozomara A. & Griffiths-Jones S. miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res. 39, D152–D157 (2011). PubMed PMC

SantaLucia J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. USA 95, 1460–1465 (1998). PubMed PMC

Rehmsmeier M., Steffen P., Höchsmann M. & Giegerich R. Fast and effective prediction of microRNA/target duplexes. RNA 10, 1507–1517 (2004). PubMed PMC

Krüger J. & Rehmsmeier M. RNAhybrid: microRNA target prediction easy, fast and flexible. Nucleic Acids Res. 34, W451–W454 (2006). PubMed PMC

Sachdeva M. et al.. p53 represses c-Myc through induction of the tumor suppressor miR-145. Proc. Natl. Acad. Sci. USA 106, 3207–3212 (2009). PubMed PMC

Shao Y., Qu Y., Dang S., Yao B. & Ji M. MiR-145 inhibits oral squamous cell carcinoma (OSCC) cell growth by targeting c-Myc and Cdk6. Cancer Cell Int. 13, 51 (2013). PubMed PMC

Xu N., Papagiannakopoulos T., Pan G., Thomson J. A. & Kosik K. S. MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell 137, 647–658 (2009). PubMed

Zhang W., Wang Q., Yu M., Wu N. & Wang H. MicroRNA-145 function as a cell growth repressor by directly targeting c-Myc in human ovarian cancer. Technol. Cancer Res. Treat. 13, 161–168 (2014). PubMed

Janssen H. L. A. et al.. Treatment of HCV infection by targeting microRNA. N. Engl. J. Med. 368, 1685–1694 (2013). PubMed

Kertesz M., Iovino N., Unnerstall U., Gaul U. & Segal E. The role of site accessibility in microRNA target recognition. Nat. Genet. 39, 1278–1284 (2007). PubMed

Hofacker I. L. Vienna RNA secondary structure server. Nucleic Acids Res. 31, 3429–3431 (2003). PubMed PMC

Liu C. et al.. CLIP-based prediction of mammalian microRNA binding sites. Nucleic Acids Res. 41, e138 (2013). PubMed PMC

Wang P. et al.. miRSponge: a manually curated database for experimentally supported miRNA sponges and ceRNAs. Database J. Biol. Databases Curation 2015 (2015). PubMed PMC

Seok H., Ham J., Jang E.-S. & Chi S. W. MicroRNA Target Recognition: Insights from Transcriptome-Wide Non-Canonical Interactions. Mol. Cells 39, 375–381 (2016). PubMed PMC

Bartel D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009). PubMed PMC

Morgado A. L., Rodrigues C. M. P. & Solá S. MicroRNA-145 Regulates Neural Stem Cell Differentiation Through the Sox2-Lin28/let-7 Signaling Pathway. Stem Cells Dayt. Ohio 34, 1386–1395 (2016). PubMed

Adammek M. et al.. MicroRNA miR-145 inhibits proliferation, invasiveness, and stem cell phenotype of an in vitro endometriosis model by targeting multiple cytoskeletal elements and pluripotency factors. Fertil. Steril. 99, 1346–1355.e5 (2013). PubMed

Huang S. et al.. miR-143 and miR-145 inhibit stem cell characteristics of PC-3 prostate cancer cells. Oncol. Rep. 28, 1831–1837 (2012). PubMed

Otaegi G., Pollock A. & Sun T. An Optimized Sponge for microRNA miR-9 Affects Spinal Motor Neuron Development in vivo. Front. Neurosci. 5, 146 (2011). PubMed PMC

Hollensen A. K., Bak R. O., Haslund D. & Mikkelsen J. G. Suppression of microRNAs by dual-targeting and clustered Tough Decoy inhibitors. RNA Biol. 10, 406–414 (2013). PubMed PMC

Scherr M. et al.. Lentivirus-mediated antagomir expression for specific inhibition of miRNA function. Nucleic Acids Res. 35, e149 (2007). PubMed PMC

Choi W.-Y., Giraldez A. J. & Schier A. F. Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430. Science 318, 271–274 (2007). PubMed

Bak R. O., Hollensen A. K., Primo M. N., Sørensen C. D. & Mikkelsen J. G. Potent microRNA suppression by RNA Pol II-transcribed ‘Tough Decoy’ inhibitors. RNA N. Y. N 19, 280–293 (2013). PubMed PMC

An Y., Wu W. & Lv A. A PCR-after-ligation method for cloning of multiple DNA inserts. Anal. Biochem. 402, 203–205 (2010). PubMed

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