This canonical maturation includes the production of the primary miRNA transcript (pri-miRNA) by RNA polymerase II or III and cleavage of the pri-miRNA by the microprocessor complex Drosha–DGCR8 (Pasha) in the nucleus [13]. A primary transcript RNA (pri-miRNA) transcribed from a miRNA gene by RNA polymerase II or III is first processed into a stem-loop structure of about 70–80 nucleotides known as precursor miRNA (pre-miRNA) by a microprocessor enzyme comprising of a double-strand (ds)-RNA-specific ribonuclease, Drosha, with the help of its binding partner DGCR8.

The resulting precursor hairpin, the pre-miRNA, is exported from the nucleus by Exportin-5–Ran-GTP [13]. Exportin-5 recognizes the pre-miRNA independently of its sequence or the loop structure. A defined length of the double-stranded stem and the 3′ overhangs are important for the successful binding to Exportin-5, ensuring the export of only correctly processed pre-miRNAs [13].

In the cytoplasm, the RNase Dicer in complex with the double-stranded RNA-binding protein TRBP cleaves the pre-miRNA hairpin to its mature length. The functional strand of the mature miRNA is loaded together with Argonaute (Ago2) proteins into the RNA-induced silencing complex (RISC), where it guides RISC to silence target mRNAs through mRNA cleavage, translational repression, or deadenylation, whereas the passenger strand is degraded [13].

Little is known about the transcriptional regulation of these intergenic miRNAs, although RNA polymerase II appears to be involved in the process [18]. This suggests that miRNAs may have active promoter regions that contain cis-regulator elements similar to coding genes. miRNA genes are currently believed to be transcribed by RNA polymerase II (Pol II), [18] although a few may be transcribed by RNA polymerase III [19]. RNA polymerase II transcribes miRNA genes, generating long primary transcripts (pri-miRNAs) [20]. Subsequently, the process to yield mature miRNAs involves two steps involving RNase III enzymes and companion double-stranded RNA-binding domain (dsRBD) proteins. There are two different classes of miRNAs with respect to transcription mechanism—those found within annotated genes (intronic miRNAs) and those found in intergenic regions of the genome (intergenic miRNAs). It is presently believed that all intronic miRNAs are co-transcribed along with their host gene; this has been shown in both expression correlation studies [21] as well as PCR-based biochemical verification [17]. Intergenic miRNAs have been postulated to come from transcripts of up to 50 kb in length, allowing for the co-transcription of neighboring miRNAs (polycistronic miRNA clusters) [21]. Identification of the method by which miRNA genes are transcribed can lead to the identification of the factors that are responsible for their regulation.

miRNAs have been associated with the regulation of differentiation, proliferation, apoptosis, and even exocytosis [22]. Volinia et al. has demonstrated that the predicted targets for the differentially expressed miRNAs are significantly enriched for protein-coding tumor suppressors and oncogenes [22]. There is also confirmation to suggest that these miRNAs function in concert with classical tumor suppressors and oncoproteins to regulate key pathways involved in cellular growth control [23, 24]. miRNA profiling has the potential not only to classify tumors but also to augur patient outcome with high accuracy, but this approach needs more validation and detailed studies by using clinical samples [25]. Jian et al. showed that profile analysis with a probe set of 201 miRNAs achieved the similar discriminative potential as traditional gene array with 8,000 mRNA probes [26]. Consequently, this would mean that classification of tumors can be achieved with a more manageable amount of data and could potentially diminish the disparity that is often seen with mRNA-based classifier systems.

Since the study of mRNA, various technologies exist that allow the investigation of the expression of either profiling of a large number of miRNAs or individual miRNAs simultaneously. In general, these observational approaches have implicated individual or groups of miRNAs in pathological or physiological processes, as a result of the detection of changes in their expression, while additional functional experiments are required to gain further knowledge of their current roles. A few miRNA profiles have been developed using a large number of single miRNA detection experiments, such as Northern blotting [14], and these technologies remain the standard against which newer profiling methods are primarily compared. Nevertheless, oligonucleotide microarray-based detection platforms, with their associated ease of use and high-throughput nature, have largely supplanted this technique [27]. Microarrays have been used for miRNAs profiling from a wide range of breast tissue types and cell lines, including formalin-fixed paraffin-embedded (FFPE) clinical samples. It is essential to note that, due to the small size of miRNAs, they are comparatively insensitive to the damage that typifies mRNAs within FFPE. Accordingly, miRNAs present an invaluable new target for studies using archival clinical samples, which can often be linked to extensive clinical background and, more importantly, follow-up data or meta-analysis [28]. Multiplex real-time RT-PCR and liquid bead-based technologies are current alternative strategies for miRNA profiling, and it is claimed that they may have a higher sensitivity and specificity [29, 30]. Methodologies based on deep sequencing of small RNA libraries obtained from tissues may also allow miRNA profiling, with the supplementary advantage that these techniques are unbiased with respect to target sequences and may permit detection of novel miRNAs [31].

The expression of miRNAs has been investigated in an extensive range of breast cancer cell lines, tissues, and clinical normal. These metadata hint toward functional roles of various miRNAs by association with cellular behavior or particular molecular markers. To gain the best insight into breast cancer, it is also essential to understand miRNA function in normal mammary gland development, and work is underway to address this question in detail [32]. A potentially powerful empirical approach is to compare miRNA expression in normal breast versus breast cancer and thereby to differentiate those miRNAs expressed at different levels. Table 8.1 lists miRNAs identified as playing a role in breast cancer and their potential targets. Iorio et al. reported for 76 breast samples diagnosed for the expression of 246 miRNAs, out of which 29 miRNAs expression levels were found to be significantly different (i.e., p <0.05) in cancer versus normal clinical tissue. The majority consistently downregulated were has-miR-10b, has-miR-125b, and has-miR-145, while has-miR-21 and has-miR-155 were upregulated, suggesting that these may act as oncogenes or tumor suppressor genes, respectively [32]. They went on to examine whether the expression profile varied according to conventional clinical aspects: ER+ /ER−, PR+ /PR−, HER2+ /HER2−, positive and negative lymph node status, presence and absence of vascular invasion, high and the low proliferation index, and ductal/lobular histopathological subtype. The majority of comparisons discovered a small number of differentially expressed miRNAs, indicating that miRNAs may have roles in defining the differences between these pathological and molecular profiles. Yet, comparison between ductal/lobular carcinomas and HER2+ and HER2− tumors did not reveal differentially expressed miRNAs.

Similarly, specific miRNA profiles have been associated with breast cancer subgroups of distinct patterns of molecular marker expression. Profiling of 204 miRNAs has been shown sufficient to allow unsupervised clustering to be used to distinguish HER2+ /ER−, HER2+ /ER−, and HER2−/ER+ breast cancers within a cohort of 20 tumors. While this in itself is not an advance, since these cancers are routinely defined using immunohistochemistry, further supervised analysis of the profiles allowed distinct miRNA subsets to be identified that distinguished HER2+ from HER2− and ER+ from ER− breast cancers, independent of other clinically important parameters. Restricted subsets of miRNAs specific to HER2 status (let-7f, let-7g, miR-107, miR-10b, miR-126, miR-154, and miR-195) and specific to ER/PR status (miR-142-5p, miR-200a, miR-205, and miR-25) have been also established [33].

The functional activity of only a few miRNAs has been practically modeled in the perspective of breast cancer, and it is clear that most of this type of work remains to be done. Potential functions of miRNAs in carcinogenesis into potential oncogenes and tumor suppressor genes have been established in regulating immune system, cell proliferation, differentiation and development, cancer and cell cycle.