A high throughput experimental approach to identify miRNA targets in human cells.
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.
Tan
Lu Ping
LP
Department of Pathology and Laboratory Medicine, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands.
Seinen
Erwin
E
Duns
Gerben
G
de Jong
Debora
D
Sibon
Ody C M
OC
Poppema
Sibrand
S
Kroesen
Bart-Jan
BJ
Kok
Klaas
K
van den Berg
Anke
A
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
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; C524941:MIRN17 microRNA, human; D035683:MicroRNAs; D008156:Luciferases