title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.
abstract: We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Motoharu
authors.initials: M
authors.lastname: Ono
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Kayo
authors.initials: K
authors.lastname: Yamada
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Fabio
authors.initials: F
authors.lastname: Avolio
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Vackar
authors.initials: V
authors.lastname: Afzal
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Dalila
authors.initials: D
authors.lastname: Bensaddek
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Angus I
authors.initials: AI
authors.lastname: Lamond
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics*; D000077192:Adenocarcinoma of Lung; D017209:Apoptosis; D060565:Argonaute Proteins / Q000235:genetics; D039642:Eukaryotic Initiation Factors / Q000235:genetics; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000494:pharmacology; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D020365:RNA Helicases; D012322:RNA Precursors / Q000235:genetics*; D016372:RNA, Antisense / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics
chemical_list: C115848:AGO1 protein, human; C466422:AGO2 protein, human; D060565:Argonaute Proteins; D039642:Eukaryotic Initiation Factors; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D012322:RNA Precursors; D016372:RNA, Antisense; D015534:Trans-Activators; D020365:RNA Helicases; C101828:UPF1 protein, human