A High Throughput Experimental Approach to Identify MiRNA Targets in Human Cells

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2009 Sep 8

PMID 19734348

Citations 60

Authors

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

Affiliations

Soon will be listed here.

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.

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References

Hammell M, Long D, Zhang L, Lee A, Carmack C, Han M . mirWIP: microRNA target prediction based on microRNA-containing ribonucleoprotein-enriched transcripts. Nat Methods. 2009; 5(9):813-9. PMC: 3092588. DOI: 10.1038/nmeth.1247. View

Edgar R, Domrachev M, Lash A . Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2001; 30(1):207-10. PMC: 99122. DOI: 10.1093/nar/30.1.207. View

Georgantas 3rd R, Hildreth R, Morisot S, Alder J, Liu C, Heimfeld S . CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control. Proc Natl Acad Sci U S A. 2007; 104(8):2750-5. PMC: 1796783. DOI: 10.1073/pnas.0610983104. View

Emmerich F, Theurich S, Hummel M, Haeffker A, Vry M, Dohner K . Inactivating I kappa B epsilon mutations in Hodgkin/Reed-Sternberg cells. J Pathol. 2003; 201(3):413-20. DOI: 10.1002/path.1454. View

Johnson S, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A . RAS is regulated by the let-7 microRNA family. Cell. 2005; 120(5):635-47. DOI: 10.1016/j.cell.2005.01.014. View

Landthaler M, Gaidatzis D, Rothballer A, Chen P, Soll S, Dinic L . Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA. 2008; 14(12):2580-96. PMC: 2590962. DOI: 10.1261/rna.1351608. View

Volinia S, Calin G, Liu C, Ambs S, Cimmino A, Petrocca F . A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006; 103(7):2257-61. PMC: 1413718. DOI: 10.1073/pnas.0510565103. View

Yeung M, Yasunaga J, Bennasser Y, Dusetti N, Harris D, Ahmad N . Roles for microRNAs, miR-93 and miR-130b, and tumor protein 53-induced nuclear protein 1 tumor suppressor in cell growth dysregulation by human T-cell lymphotrophic virus 1. Cancer Res. 2008; 68(21):8976-85. PMC: 2596768. DOI: 10.1158/0008-5472.CAN-08-0769. View

Sylvestre Y, De Guire V, Querido E, Mukhopadhyay U, Bourdeau V, Major F . An E2F/miR-20a autoregulatory feedback loop. J Biol Chem. 2006; 282(4):2135-43. DOI: 10.1074/jbc.M608939200. View

10.

Lewis B, Burge C, Bartel D . Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005; 120(1):15-20. DOI: 10.1016/j.cell.2004.12.035. View

11.

Beitzinger M, Peters L, Zhu J, Kremmer E, Meister G . Identification of human microRNA targets from isolated argonaute protein complexes. RNA Biol. 2007; 4(2):76-84. DOI: 10.4161/rna.4.2.4640. View

12.

Bonci D, Coppola V, Musumeci M, Addario A, Giuffrida R, Memeo L . The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nat Med. 2008; 14(11):1271-7. DOI: 10.1038/nm.1880. View

13.

Tsang W, Kwok T . Let-7a microRNA suppresses therapeutics-induced cancer cell death by targeting caspase-3. Apoptosis. 2008; 13(10):1215-22. DOI: 10.1007/s10495-008-0256-z. View

14.

Mott J, Kobayashi S, Bronk S, Gores G . mir-29 regulates Mcl-1 protein expression and apoptosis. Oncogene. 2007; 26(42):6133-40. PMC: 2432524. DOI: 10.1038/sj.onc.1210436. View

15.

Zhu S, Si M, Wu H, Mo Y . MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1). J Biol Chem. 2007; 282(19):14328-36. DOI: 10.1074/jbc.M611393200. View

16.

Zhang L, Ding L, Cheung T, Dong M, Chen J, Sewell A . Systematic identification of C. elegans miRISC proteins, miRNAs, and mRNA targets by their interactions with GW182 proteins AIN-1 and AIN-2. Mol Cell. 2007; 28(4):598-613. PMC: 2186060. DOI: 10.1016/j.molcel.2007.09.014. View

17.

Liang Y, Ridzon D, Wong L, Chen C . Characterization of microRNA expression profiles in normal human tissues. BMC Genomics. 2007; 8:166. PMC: 1904203. DOI: 10.1186/1471-2164-8-166. View

18.

Karginov F, Conaco C, Xuan Z, Schmidt B, Parker J, Mandel G . A biochemical approach to identifying microRNA targets. Proc Natl Acad Sci U S A. 2007; 104(49):19291-6. PMC: 2148283. DOI: 10.1073/pnas.0709971104. View

19.

Landais S, Landry S, Legault P, Rassart E . Oncogenic potential of the miR-106-363 cluster and its implication in human T-cell leukemia. Cancer Res. 2007; 67(12):5699-707. DOI: 10.1158/0008-5472.CAN-06-4478. View

20.

Hossain A, Kuo M, Saunders G . Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA. Mol Cell Biol. 2006; 26(21):8191-201. PMC: 1636750. DOI: 10.1128/MCB.00242-06. View