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 ∼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.

MYCN/c-MYC-induced microRNAs repress coding gene networks associated with poor outcome in MYCN/c-MYC-activated tumors

“…In addition, several predicted targets defined by this strategy, such as TGFBR2, ATXN1 and HIPK3, have already been reported as direct targets of miR-20a, miR-19a and miR-92a, respectively (Volinia et al, 2006;Landais et al, 2007;Lee et al, 2008). We also compared our target predictions for miR-20a in neuroblastoma to a set of validated miR-20a targets obtained by ribonucleoprotein immunoprecipitation-gene Chip in Hodgkin lymphoma cells (Tan et al, 2009). From the 37 miR20a 8mer targets that we identified, 14 were reported by Tan et al (2009) (Fisher's exact test, Po0.001).…”

“…We also compared our target predictions for miR-20a in neuroblastoma to a set of validated miR-20a targets obtained by ribonucleoprotein immunoprecipitation-gene Chip in Hodgkin lymphoma cells (Tan et al, 2009). From the 37 miR20a 8mer targets that we identified, 14 were reported by Tan et al (2009) (Fisher's exact test, Po0.001). These results indicate that, although some of the predicted targets could be indirect, our miRNA target identification strategy definitely selects a substantial number of direct miRNA target genes.…”

Multidrug Resistance-associated Protein-1 (MRP-1)-dependent Glutathione Disulfide (GSSG) Efflux as a Critical Survival Factor for Oxidant-enriched Tumorigenic Endothelial Cells

“…All siRNA SMARTpool reagents were from Dharmacon RNA Technologies (Lafayette, CO). For controls, siControl non-targeting siRNA pool (mixture of four siRNAs, designed to have Ն4 mismatches with the corresponding gene) was used (42). DharmaFECT 1 transfection reagent (Dharmacon RNA Technologies) was used to transfect cells with the mentioned siRNAs (Dharmacon RNA Technologies) for 72 h per the manufacturer's instructions.…”

Dicer Knockdown Inhibits Endothelial Cell Tumor Growth via MicroRNA 21a-3p Targeting of Nox-4

“…Cells were transfected as described above with control and dicer siRNA for 72 h. After 72 h, cells (3 wells/sample) were washed with ice-cold phosphate-buffered saline (pH 7.4) and lysed with lysis buffer (150 mM KCl, 25 mM Tris-HCl, 5 mM EDTA, 0.5% IgePal, 1 mM PMSF, 1ϫ protease inhibitor) as described previously (21). Protein concentration was determined using the BCA protein assay kit (Pierce).…”

“…Immu- noprecipitated complexes were washed three times with lysis buffer (centrifugation at 2500 ϫ g at 4°C for 5 min). For Western blot, samples were subjected to SDS/PAGE after reduction with 1 M DTT (21,22). RNA were isolated using TRIzol and glycogen (Invitrogen) (21).…”