title: A high throughput experimental approach to identify miRNA targets in human cells.
abstract: The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation-gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that approximately 40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5'-UTRs, coding regions and 3'-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3'-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3'-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner.
authors.affiliation: Department of Pathology and Laboratory Medicine, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands.
authors.forename: Lu Ping
authors.initials: LP
authors.lastname: Tan
authors.forename: Erwin
authors.initials: E
authors.lastname: Seinen
authors.forename: Gerben
authors.initials: G
authors.lastname: Duns
authors.forename: Debora
authors.initials: D
authors.lastname: de Jong
authors.forename: Ody C M
authors.initials: OC
authors.lastname: Sibon
authors.forename: Sibrand
authors.initials: S
authors.lastname: Poppema
authors.forename: Bart-Jan
authors.initials: BJ
authors.lastname: Kroesen
authors.forename: Klaas
authors.initials: K
authors.lastname: Kok
authors.forename: Anke
authors.initials: A
authors.lastname: van den Berg
mesh_terms: D060565:Argonaute Proteins; D001665:Binding Sites; D002460:Cell Line; D015852:Eukaryotic Initiation Factor-2 / Q000302:isolation & purification / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D047468:Immunoprecipitation; D008156:Luciferases / Q000235:genetics; D035683:MicroRNAs / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; C524941:MIRN17 microRNA, human; D035683:MicroRNAs; D008156:Luciferases