A high throughput experimental approach to identify miRNA targets in human cells

Lu Ping Tan, Erwin Seinen, Gerben Duns, Debora de Jong, Ody C. M. Sibon, Sibrand Poppema, Bart-Jan Kroesen, Klaas Kok, Anke van den Berg*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

90 Citaten (Scopus)

Samenvatting

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 similar to 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.

Originele taal-2English
Artikelnummer137
Aantal pagina's12
TijdschriftNucleic Acids Research
Volume37
Nummer van het tijdschrift20
DOI's
StatusPublished - nov-2009

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