A high throughput experimental approach to identify miRNA targets in human cells
Nucleic acids research, 2009•academic.oup.com
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∼ 40% of these gene …
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∼ 40% of these gene …
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 ∼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.
Oxford University Press