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

Suvi-Katri Leivonen (Corresponding Author), Rami Mäkelä, Päivi Östling, Pekka Kohonen, Saija Haapa-Paananen, Kristine Kleivi, E. Enerly, Anna Aakula, K. Hellström, Niko Sahlberg, V.N. Kristensen, A.-L. Børresen-Dale, Petri Saviranta, Merja Perälä, Olli Kallioniemi

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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.
Original languageEnglish
Pages (from-to)3926-3936
Number of pages11
JournalOncogene
Volume28
DOIs
Publication statusPublished - 2009
MoE publication typeA1 Journal article-refereed

Fingerprint

Protein Array Analysis
MicroRNAs
Estrogen Receptors
Breast Neoplasms
Cell Line
Estrogens
Technology
Proteins
3' Untranslated Regions
Transcriptome
Small Interfering RNA
Transfection
Down-Regulation
Western Blotting
Growth
Genes

Cite this

Leivonen, S-K., Mäkelä, R., Östling, P., Kohonen, P., Haapa-Paananen, S., Kleivi, K., ... Kallioniemi, O. (2009). Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines. Oncogene, 28, 3926-3936. https://doi.org/10.1038/onc.2009.241
Leivonen, Suvi-Katri ; Mäkelä, Rami ; Östling, Päivi ; Kohonen, Pekka ; Haapa-Paananen, Saija ; Kleivi, Kristine ; Enerly, E. ; Aakula, Anna ; Hellström, K. ; Sahlberg, Niko ; Kristensen, V.N. ; Børresen-Dale, A.-L. ; Saviranta, Petri ; Perälä, Merja ; Kallioniemi, Olli. / Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines. In: Oncogene. 2009 ; Vol. 28. pp. 3926-3936.
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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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Leivonen, S-K, Mäkelä, R, Östling, P, Kohonen, P, Haapa-Paananen, S, Kleivi, K, Enerly, E, Aakula, A, Hellström, K, Sahlberg, N, Kristensen, VN, Børresen-Dale, A-L, Saviranta, P, Perälä, M & Kallioniemi, O 2009, 'Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines', Oncogene, vol. 28, pp. 3926-3936. https://doi.org/10.1038/onc.2009.241

Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines. / Leivonen, Suvi-Katri (Corresponding Author); Mäkelä, Rami; Östling, Päivi; Kohonen, Pekka; Haapa-Paananen, Saija; Kleivi, Kristine; Enerly, E.; Aakula, Anna; Hellström, K.; Sahlberg, Niko; Kristensen, V.N.; Børresen-Dale, A.-L.; Saviranta, Petri; Perälä, Merja; Kallioniemi, Olli.

In: Oncogene, Vol. 28, 2009, p. 3926-3936.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines

AU - Leivonen, Suvi-Katri

AU - Mäkelä, Rami

AU - Östling, Päivi

AU - Kohonen, Pekka

AU - Haapa-Paananen, Saija

AU - Kleivi, Kristine

AU - Enerly, E.

AU - Aakula, Anna

AU - Hellström, K.

AU - Sahlberg, Niko

AU - Kristensen, V.N.

AU - Børresen-Dale, A.-L.

AU - Saviranta, Petri

AU - Perälä, Merja

AU - Kallioniemi, Olli

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

U2 - 10.1038/onc.2009.241

DO - 10.1038/onc.2009.241

M3 - Article

VL - 28

SP - 3926

EP - 3936

JO - Oncogene

JF - Oncogene

SN - 0950-9232

ER -