**4.2. Role of RNAi technology in breast cancer therapy**

In the context of breast cancer therapy, target genes can be silenced by inhibiting miRNA with drugs or by substituting exogenous miRNA. This type of inhibition therapy is deemed useful when tumor cells are known to overexpress the target miRNA. The therapy involves the introduction of single-stranded, synthetic, RNAs that effectively act as miRNA antagonists. In this regard, the inhibition of miRNA is analogous to antisense inhibition. In contrast, cancer be treated by replacing miRNA when the target miRNA is deactivated or repressed, a common feature of cancer. This strategy involves introducing double stranded synthetic miRNAs into cells, which will mimic the actions of the target miRNAs. They bind to the target gene and initiate mRNA degradation which silences the target gene. miRNA mimics are more straightforward to use than siRNA, because the sequences are nearly identical to the endogenous miRNA [6].

In an alternative approach, siRNAs can be produced through gene expression or by chemical synthesis. The strategy governing chemical synthesis is to create synthetic RNAs that can be introduced into cells by a number of methods. The gene expression strategy can produce siRNA using expression constructs, including viral vectors and plasmids, that can be transcribed inside the cell and express short hairpin RNA (shRNAs) that are the precursors of siRNAs. When siRNAs are chemically synthesized this method provides fine control over the purity and quantity of the siRNAs produced. Furthermore, the chemical structure can be modified to improve the stability of the product, which is important when the delivery technique has to be considered. Synthetic siRNAs can also incorporate fluorescence labels using high-resolution fluorescence microscopy to analyze siRNA and its localization in cancer cells [6]. After the introduction of the siRNA into the cell, gene silencing is initiated, a process directed by the endogenous RNAi machinery. Within the cell, duplex siRNAs are incorporated into the RNAi pathway. The antisense strands are loaded onto the RNA induced silencing complex (RISC), which acts as a guide to permit the recognition of complementary mRNAs. Once the target sequence is identified, AGO2 protein, which is a constituent of the RISC complex, cleaves the mRNA decreasing the protein levels produced by silenced gene. The main advantage of using siRNAs vs. conventional drug therapy is that they exhibit very low toxicity and a high degree of specificity. However, non-specific targets can be affected mainly because the miRNA-like activity of the siRNAs can act at the seed-like sequence of the 5′ end leading to a stimulation of the innate immune system induced by dsRNA [6].

It should be recalled that both siRNAs and mimetics engage with the same related RNAi pathways and interact with the RISC complex. Although siRNA and miRNA appear to be similar in all respects profound differences have been identified. For example, it is known that miRNAs are reproduced as primary miRNA transcripts (pri-miRs) through the activity of miR genes. The controlling nuclear complex partially cleaves the miRNA and resulting stem loop pre-miR is translocated from the nucleus to the cell cytoplasm where it is acted on by Dicer, thus transforming it into dsRNA. The dsRNA now consist of a mature or active strand or may incorporate an inactive "passenger strand". miRNA which is mature then interacts with the RISC complex to silence genes. Short dsRNAs, termed siRNAs, are assemblies of 21- to 23 base pairs, with typically 2 nucleotide overhangs at the 3′ ends, although numerous variations have been detected in their length and the degree of overhang. Structurally, they contain an active guide strand and an inactive passenger strand and can be considered 'exogenous RNAs' that can engage with endogenous RNAi pathways. It should be noted that miRNA often has defective complementarity, usually because of changes in the seed sequence at the 5′ end [6]. This finding stands in stark contrast to siRNA and its target mRNA, where there is a perfect fit (full complementarity) between the two composite molecules.-
