title: Rapid generation of microRNA sponges for microRNA inhibition.
abstract: MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.
authors.affiliation: Department of Pathology and Medical Biology, University Medical Center Groningen, Groningen, The Netherlands. j.l.kluiver@umcg.nl
authors.forename: Joost
authors.initials: J
authors.lastname: Kluiver
authors.forename: Johan H
authors.initials: JH
authors.lastname: Gibcus
authors.forename: Chris
authors.initials: C
authors.lastname: Hettinga
authors.forename: Annelies
authors.initials: A
authors.lastname: Adema
authors.forename: Mareike K S
authors.initials: MK
authors.lastname: Richter
authors.forename: Nancy
authors.initials: N
authors.lastname: Halsema
authors.forename: Izabella
authors.initials: I
authors.lastname: Slezak-Prochazka
authors.forename: Ye
authors.initials: Y
authors.lastname: Ding
authors.forename: Bart-Jan
authors.initials: BJ
authors.lastname: Kroesen
authors.forename: Anke
authors.initials: A
authors.lastname: van den Berg
mesh_terms: D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology; D016372:RNA, Antisense / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: C549214:MIRN19A microRNA, human; D035683:MicroRNAs; D016372:RNA, Antisense