RNA abundance and DNA copy number are routinely measured in high-throughput using microarray and next-generation sequencing (NGS) technologies, and the attributes of different platforms have been extensively analyzed. Recently, the application of both microarrays and NGS has expanded to include microRNAs (miRNAs), but the relative performance of these methods has not been rigorously characterized. We analyzed three biological samples across six miRNA microarray platforms and compared their hybridization performance. We examined the utility of these platforms, as well as NGS, for the detection of differentially expressed miRNAs. We then validated the results for 89 miRNAs by real-time RT-PCR and challenged the use of this assay as a ''gold standard.'' Finally, we implemented a novel method to evaluate false-positive and false-negative rates for all methods in the absence of a reference method.

MicroRNAs (miRNAs) show differential expression across breast cancer subtypes, and have both oncogenic and tumour-suppressive roles. Here we report the miRNA expression profiles of 1,302 breast tumours with matching detailed clinical annotation, long-term follow-up and genomic and messenger RNA expression data. This provides a comprehensive overview of the quantity, distribution and variation of the miRNA population and provides information on the extent to which genomic, transcriptional and post-transcriptional events contribute to miRNA expression architecture, suggesting an important role for post-transcriptional regulation. The key clinical parameters and cellular pathways related to the miRNA landscape are characterized, revealing context-dependent interactions, for example with regards to cell adhesion and Wnt signalling. Notably, only prognostic miRNA signatures derived from breast tumours devoid of somatic copy-number aberrations (CNA-devoid) are consistently prognostic across several other subtypes and can be validated in external cohorts. We then use a data-driven approach to seek the effects of miRNAs associated with differential co-expression of mRNAs, and find that miRNAs act as modulators of mRNA-mRNA interactions rather than as on-off molecular switches. We demonstrate such an important modulatory role for miRNAs in the biology of CNA-devoid breast cancers, a common subtype in which the immune response is prominent. These findings represent a new framework for studying the biology of miRNAs in human breast cancer.

Vg1 mRNA translocation to the vegetal cortex of Xenopus oocytes requires intact microtubules, and a 3 UTR cis-acting element (termed VLE), which also mediates sequence-specific binding of several proteins. One protein, the 69-kD Vg1 RBP, associates Vg1 RNA to microtubules in vitro. Here we show that Vg1 RBP-binding sites correlate with vegetal localization. Purification and cloning of Vg1 RBP revealed five RNA-binding motifs: four KH and one RRM domains. Surprisingly, Vg1 RBP is highly homologous to the zipcode binding protein implicated in the microfilament-mediated localization of ␤ actin mRNA in fibroblasts. These data support Vg1 RBP's direct role in vegetal localization and suggest the existence of a general, evolutionarily conserved mechanism for mRNA targeting. Received January 21, 1998; revised version accepted March 25, 1998. Intracellular RNA localization leads to asymmetric protein synthesis, a necessary step in the process of pattern formation during early embryogenesis in many species (for review, see St. Johnston 1995;Gavis 1997). In Xenopus oocytes, morphological and molecular differences between the animal and vegetal hemispheres help define the primary axis around which subsequent development proceeds. Two temporally distinct pathways have been identified for vegetal RNA localization. The early pathway facilitates localization of several RNAs found to be localized during stages I-II of oogenesis (Xcat-2, Xlsirts, Xwnt-11), appears to be microtubule-independent, and is correlated with the migration of the mitochondrial cloud to a small region at the vegetal pole (Kloc et al. 1993;Mosquera et al. 1993;Forristall et al. 1995;Kloc and Etkin 1995;Zhou and King 1996a). The second pathway occurs during late stage III-early stage IV, requires intact microtubules, and targets RNA to a tight shell along the entire vegetal cortex (Melton 1987;Yisraeli and Melton 1988;Yisraeli et al. 1990). So far, only one mRNA, Vg1, is known to localize via the second pathway in vivo, although Xcat-2 RNA can employ this pathway when injected into stage III oocytes (Zhou and King 1996b). VegT/Xombi/Apod/Brat RNA is also localized to the vegetal cortex in a manner resembling the second pathway, but it is not yet known whether this localization requires intact microtubules; furthermore, it is released from the cortex in stage V/VI oocytes, significantly earlier than is Vg1 RNA (Lustig et al. 1996;Stennard et al. 1996;Zhang and King 1996). Cis-acting elements mediating one or the other pathway have been mapped, by deletion analysis, to extensive regions of the 3Ј UTRs (Mowry and Melton 1992; Zhou and King 1996a,b; Gautreau et al. 1997). Trans-acting factors that bind these regions and may provide a link between the RNA and components of the cytoskeleton have been characterized only preliminarily, and the connection between these interactons and localization is just beginning to be defined (Schwartz et al. 1992;Mowry 1996;Deshler et al. 1997).Vg1 RNA-binding protein (RBP) is a 69-kD oocyte protein that binds specifically t...

The telomeric amplicon at 8p12 is common in oestrogen receptor-positive (ER+) breast cancers. Array-CGH and expression analyses of 1172 primary breast tumours revealed that ZNF703 was the single gene within the minimal amplicon and was amplified predominantly in the Luminal B subtype. Amplification was shown to correlate with increased gene and protein expression and was associated with a distinct expression signature and poor clinical outcome. ZNF703 transformed NIH 3T3 fibroblasts, behaving as a classical oncogene, and regulated proliferation in human luminal breast cancer cell lines and immortalized human mammary epithelial cells. Manipulation of ZNF703 expression in the luminal MCF7 cell line modified the effects of TGFβ on proliferation. Overexpression of ZNF703 in normal human breast epithelial cells enhanced the frequency of in vitro colony-forming cells from luminal progenitors. Taken together, these data strongly point to ZNF703 as a novel oncogene in Luminal B breast cancer.