6 Journal of Aging Research Targeting onco-miRs Anti-miRs miRNA sponge miR-mask mRNA AAAAAA miR:anti-miRs miR:mRNA sponge mRNA:miR-mask mRNA: AAAAAAAA Figure 3: miRNA-based molecular cancer therapy for oncogenic miRNAs. The cancer therapies include anti-miRNA oligonucleotides (anti- miRs), miRNA sponges, and miRNA masking. miRNAs. Therefore, a logical approach of silencing miRNAs is to use a nucleic acid that is antisense to the miRNA [84, 85]. These anti-miRNA oligonucleotides (AMOs) block the interactions between miRNAs and their target mRNAs by competition. Thus, the anti-miRNA oligonucleotides knockdown the oncogenic properties of the miRNA resulting in cancer suppression and decreased cancer progression. Studies targeting miR-21 represent one of the ﬁrst examples of inhibiting cancer development by downregulating an oncogenic miRNA. miR-21 is overexpressed in most tumor types and acts by targeting many tumor suppressor genes related to proliferation, apoptosis, and invasion including the following genes: programmed cell death 4 (PDCD4) [86–88]; tropomyosin 1 (Tpm1) [89]; PTEN [90, 91]; ras homolog gene family, member B (RHOB) [92]; polymerase (DNA-directed), delta 4 (POLD4) [93]. Si and colleagues have knocked down miR-21 expression using an anti-miR-21 oligonucleotide transfected into MCF-7 breast cancer cells, and they demonstrated that the anti-miR-21 oligonucleotide suppressed both cell growth and tumor growth in a xenograft mouse model by increasing apoptosis and decreasing cell proliferation [94]. A modiﬁed AMO approach has recently been described in which multiple antisense units are engineered into a single unit that is able to simultaneously silence multiple miRNAs. For example, the multiple-target AMO targeting miR-21, miR-155, and miR- 17-5p has a greater inhibitory eﬀect on cell growth in MCF-7 cells when compared to single- target AMOs or a combination of these single-target AMOs [95]. The multiple-target AMO approach may have a broad application in human tumors. 4.2. miRNA Sponges. miRNA sponges are transcripts that contain multiple tandem-binding sites to a miRNA of interest, therefore preventing the interaction between the miRNA and its endogenous targets. Ebert et al. (2007) [96] engineered such molecules by inserting a bulge between the miRNA-binding sites at the position normally cleaved by Argonaute 2, thereby enabling stable association of miRNA sponges with microribonucleoprotein complexes loaded with the corresponding miRNA. In addition, they speciﬁcally designed sponges with a complementary heptameric seed so that a single sponge can be used to eﬀectively repress an entire miRNA seed family. These miRNA sponges can derepress miRNA targets as strongly as chemically modiﬁed AMOs in vitro. miRNA sponges have been applied to inhibit miRNA activity in Drosophila [97]. However, the eﬃcacy of these stably expressed sponges in applications needs to be further evaluated. 4.3. miRNA Masking. A miRNA-mask is a gene-speciﬁc strategy developed by Xiao et al. (2007). miR-masks consist of single-stranded 2 -O-methyl-modiﬁed antisense oligonu- cleotides that are fully complementary to predicted miRNA binding sites in the 3 -UTR of the target mRNA. These modiﬁed oligonucleotides can form complementary duplex fragments with the target mRNA with higher aﬃnity. In this study, Xiao and colleagues designed miR-masks complemen- tary to HCN2 and HCN4 mRNA to prevent the repressive actions of miR-1 and miR-133 on protein expression of these genes [98]. The disadvantage of this gene-speciﬁc strategy is the limited scope (one target) for therapeutic purposes. 4.4. Restoring Tumor Suppressor miRNA Expression. For TS-miRs, which promote cancer when downregulated, small synthetic oligonucleotides that mimic endogenous mature miRNA molecules (designated miRNA mimics) restore expression of TS-miRs, thereby inducing cell death and blocking proliferation [99, 100]. The concept of miRNA replacement therapy is perhaps best exempliﬁed by the let-7 miRNA family. Let-7 is underexpressed in nonsmall cell lung cancer relative to normal lung tissue, which inversely correlates with the expression of the Ras oncoprotein, suggesting that let-7 negatively regulates the Ras oncoprotein