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Profiling of estrogen-regulated microRNAs in breast cancer cells.
(Cecilia Williams)ORCID iD: 0000-0002-0602-2062
2014 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 84, article id e51285Article in journal (Refereed) Published
Abstract [en]

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Place, publisher, year, edition, pages
JoVE , 2014. no 84, article id e51285
Keywords [en]
Breast cancer; Estrogen; Estrogen receptor; Issue 84; Medicine; Microarray; MicroRNA; qPCR
National Category
Other Medical Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-206376DOI: 10.3791/51285ISI: 000348604100060PubMedID: 24637950Scopus ID: 2-s2.0-84904540817OAI: oai:DiVA.org:kth-206376DiVA, id: diva2:1092207
Funder
NIH (National Institute of Health), R01CA172437
Note

QC 20170503

Available from: 2017-05-02 Created: 2017-05-02 Last updated: 2017-11-29Bibliographically approved

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