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Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines

Oncogene volume 28, pages 3926–3936 (2009)Cite this article

Abstract

Predicting the impact of microRNAs (miRNAs) on target proteins is challenging because of their different regulatory effects at the transcriptional and translational levels. In this study, we applied a novel protein lysate microarray (LMA) technology to systematically monitor for target protein levels after high-throughput transfections of 319 pre-miRs into breast cancer cells. We identified 21 miRNAs that downregulated the estrogen receptor-α (ERα), as validated by western blotting and quantitative real time–PCR, and by demonstrating the inhibition of estrogen-stimulated cell growth. Five potent ERα-regulating miRNAs, miR-18a, miR-18b, miR-193b, miR-206 and miR-302c, were confirmed to directly target ERα in 3′-untranslated region reporter assays. The gene expression signature that they repressed highly overlapped with that of a small interfering RNA against ERα, and across all the signatures tested, was most closely associated with the repression of known estrogen-induced genes. Furthermore, miR-18a and miR-18b showed higher levels of expression in ERα-negative as compared with ERα-positive clinical tumors. In summary, we present systematic and direct functional evidence of miRNAs inhibiting ERα signaling in breast cancer, and demonstrate the high-throughput LMA technology as a novel, powerful technique in determining the relative impact of various miRNAs on key target proteins and associated cellular processes and pathways.

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Acknowledgements

The technical assistance of Pauliina Toivonen is gratefully acknowledged. We thank Agilent Technologies for their support on the miRNA expression arrays, and Hilde Johnsen (Department of Genetics, Norwegian Radium Hospital) for laboratory handling of miRNA expression arrays on tumors. We are grateful to the Department of Pathology, Norwegian Radium Hospital, for providing ER-IHC. This study was supported by the Academy of Finland Centre of Excellence on Translational Genome-Scale Biology, Academy of Finland postdoctoral researcher's grant (to SKL), Sigrid Juselius Foundation, Finnish Cancer Society, EU-FP6 projects RIGHT (LSHB-CT-2004-005276) and Moltools (LSHG-CT-2004-5033155), and EU-FP7 project ProspeR (HEALTH-F2-2007-201438).

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Authors and Affiliations

  1. Medical Biotechnology, VTT Technical Research Centre of Finland, and Centre for Biotechnology, University of Turku, Turku, Finland

    S-K Leivonen, R Mäkelä, P Östling, P Kohonen, S Haapa-Paananen, K Kleivi, A Aakula, K Hellström, N Sahlberg, P Saviranta, M Perälä & O Kallioniemi

  2. Department of Genetics, Institute for Cancer Research, Norwegian Radiumhospital, Rikshospitalet University Hospital, Oslo, Norway

    K Kleivi, E Enerly, V N Kristensen & A-L Børresen-Dale

  3. Division of The Norwegian Radiumhospital, Faculty of Medicine, University of Oslo, Oslo, Norway

    E Enerly & A-L Børresen-Dale

  4. Division of AHUS, Faculty of Medicine, University of Oslo, Oslo, Norway

    V N Kristensen

  5. Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland

    O Kallioniemi

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  1. S-K Leivonen
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  2. R Mäkelä
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Correspondence to S-K Leivonen.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Leivonen, SK., Mäkelä, R., Östling, P. et al. Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines. Oncogene 28, 3926–3936 (2009). https://doi.org/10.1038/onc.2009.241

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