title: Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines.
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.
authors.affiliation: Medical Biotechnology, VTT Technical Research Centre of Finland, and Centre for Biotechnology, University of Turku, Turku, Finland. suvi-katri.leivonen@vtt.fi
authors.forename: S-K
authors.initials: SK
authors.lastname: Leivonen
authors.forename: R
authors.initials: R
authors.lastname: Mäkelä
authors.forename: P
authors.initials: P
authors.lastname: Ostling
authors.forename: P
authors.initials: P
authors.lastname: Kohonen
authors.forename: S
authors.initials: S
authors.lastname: Haapa-Paananen
authors.forename: K
authors.initials: K
authors.lastname: Kleivi
authors.forename: E
authors.initials: E
authors.lastname: Enerly
authors.forename: A
authors.initials: A
authors.lastname: Aakula
authors.forename: K
authors.initials: K
authors.lastname: Hellström
authors.forename: N
authors.initials: N
authors.lastname: Sahlberg
authors.forename: V N
authors.initials: VN
authors.lastname: Kristensen
authors.forename: A-L
authors.initials: AL
authors.lastname: Børresen-Dale
authors.forename: P
authors.initials: P
authors.lastname: Saviranta
authors.forename: M
authors.initials: M
authors.lastname: Perälä
authors.forename: O
authors.initials: O
authors.lastname: Kallioniemi
mesh_terms: D020413:3' Untranslated Regions; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D002474:Cell-Free System / Q000378:metabolism; D015536:Down-Regulation; D047628:Estrogen Receptor alpha / Q000096:biosynthesis*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D012334:RNA, Neoplasm / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D020413:3' Untranslated Regions; D047628:Estrogen Receptor alpha; D035683:MicroRNAs; D012334:RNA, Neoplasm