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Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells
Published on: February 21, 2014
Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell
S-K Leivonen1, R Mäkelä, P Ostling
1Medical Biotechnology, VTT Technical Research Centre of Finland, and Centre for Biotechnology, University of Turku, Turku, Finland. suvi-katri.leivonen@vtt.fi
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-alpha (ERalpha), as validated by western blotting and quantitative real time-PCR, and by demonstrating the inhibition of estrogen-stimulated cell growth. Five potent ERalpha-regulating miRNAs, miR-18a, miR-18b, miR-193b, miR-206 and miR-302c, were confirmed to directly target ERalpha in 3'-untranslated region reporter assays. The gene expression signature that they repressed highly overlapped with that of a small interfering RNA against ERalpha, 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 ERalpha-negative as compared with ERalpha-positive clinical tumors. In summary, we present systematic and direct functional evidence of miRNAs inhibiting ERalpha 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.
Insights
This study reveals specific microRNAs (miRNAs) that inhibit estrogen receptor-alpha (ERalpha) signaling in breast cancer. Novel microarray technology identified potent miRNAs regulating ERalpha, offering new therapeutic targets.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- Predicting microRNA (miRNA) effects on target proteins is complex due to transcriptional and translational regulation.
- Estrogen receptor-alpha (ERalpha) is a key driver in many breast cancers, making its regulation a critical therapeutic target.
Purpose of the Study:
- To systematically identify miRNAs that regulate ERalpha levels and function in breast cancer cells.
- To validate the direct targeting of ERalpha by specific miRNAs.
- To assess the utility of protein lysate microarray (LMA) technology for high-throughput miRNA screening.
Main Methods:
- High-throughput screening using a novel protein lysate microarray (LMA) with 319 pre-miRs in breast cancer cells.
- Validation of miRNA targets using western blotting, quantitative real-time PCR, and functional assays (estrogen-stimulated cell growth inhibition).
- 3'-untranslated region (3'-UTR) reporter assays to confirm direct miRNA targeting of ERalpha.
- Comparison of miRNA-induced gene expression signatures with ERalpha small interfering RNA (siRNA) and estrogen-induced genes.
Main Results:
- Identified 21 miRNAs that downregulate ERalpha protein levels.
- Confirmed five potent ERalpha-regulating miRNAs (miR-18a, miR-18b, miR-193b, miR-206, miR-302c) that directly target ERalpha.
- Demonstrated that these miRNAs inhibit estrogen-stimulated breast cancer cell growth.
- Showed that miRNA-repressed gene signatures closely resemble those of ERalpha knockdown and are associated with known estrogen-induced genes.
- Observed higher miR-18a and miR-18b expression in ERalpha-negative tumors compared to ERalpha-positive tumors.
Conclusions:
- Provided systematic and direct functional evidence for miRNAs inhibiting ERalpha signaling in breast cancer.
- Established high-throughput LMA as a powerful technique for assessing miRNA impact on target proteins and cellular pathways.
- Highlighted specific miRNAs as potential therapeutic agents or biomarkers for ERalpha-positive and ERalpha-negative breast cancers.
