- freely available
Pharmaceuticals 2013, 6(10), 1195-1220; doi:10.3390/ph6101195
Published: 30 September 2013
Abstract: The discovery of small RNA molecules with the capacity to regulate messenger RNA (mRNA) stability and translation (and consequently protein synthesis) has revealed an additional level of post-transcriptional gene control. MicroRNAs (miRNAs), an evolutionarily conserved class of small noncoding RNAs that regulate gene expression post-transcriptionally by base pairing to complementary sequences in the 3' untranslated regions of target mRNAs, are part of this modulatory RNA network playing a pivotal role in cell fate. Functional studies indicate that miRNAs are involved in the regulation of almost every biological pathway, while changes in miRNA expression are associated with several human pathologies, including cancer. By targeting oncogenes and tumor suppressors, miRNAs have the ability to modulate key cellular processes that define the cell phenotype, making them highly promising therapeutic targets. Over the last few years, miRNA-based anti-cancer therapeutic approaches have been exploited, either alone or in combination with standard targeted therapies, aiming at enhancing tumor cell killing and, ideally, promoting tumor regression and disease remission. Here we provide an overview on the involvement of miRNAs in cancer pathology, emphasizing the mechanisms of miRNA regulation. Strategies for modulating miRNA expression are presented and illustrated with representative examples of their application in a therapeutic context.
Over the last decade, miRNAs have emerged as important players in the highly complex world of gene regulation. A large number of studies involving transcriptomic, proteomic and bioinformatic approaches indicate that these small RNA molecules can regulate over 30% of all protein-coding genes and play a pivotal role in the most basic cellular processes—such as embryonic development, cell differentiation, metabolism, proliferation and cell death—in a wide range of invertebrate and vertebrate organisms, including humans [1,2,3,4]. Widespread influence of miRNAs is also observed in different physiological responses, like cardiovascular development , stem cell differentiation , immune response , insulin secretion  and anti-viral defense . Since the miRNA milieu has a broad effect over diverse genetic and molecular pathways, it is not surprising that abnormal miRNA expression has been associated with several human diseases, including cardiovascular and neurological disorders [10,11], diabetes  and cancer . In this review, we briefly describe the miRNA biogenesis and miRNA-mediated gene silencing mechanisms and discuss the current knowledge on how miRNAs can act as oncogenes and tumor suppressors. We also address recent advances on the application of miRNA-based therapeutic approaches to cancer.
2. MiRNA Biogenesis and Gene Silencing Mechanisms
The synthesis of miRNAs from their initial chromosome encryption to the final mature form is a highly regulated stepwise process that takes place in the cell nucleus and cytoplasm, and may interact with other important cellular functions, including splicing. In the biologically predominant canonical pathway, miRNAs are processed from 5'-capped and 3'-polyadenylated precursor molecules, designated primary miRNAs (pri-miRNAs), which are subsequently cleaved by a microprocessor complex that includes the RNase III enzyme Drosha and the double stranded (ds) RNA-binding protein DGCR8, to produce a 70-nucleotide (nt) hairpin-structured miRNA precursor (pre-miRNA). The pre-miRNA is exported from the nucleus to the cytoplasm via the transporter exportin-5, in a GTP-dependent process, where it is cleaved by a multiprotein complex that includes the endonuclease Dicer and the RNA-binding protein TAR (TRBP), yielding a miRNA duplex of approximately 21 to 23 nucleotides, with 2-nt overhangs at the 3' ends. In addition to the biologically prevalent canonical pathway, where miRNA precursor molecules are produced by the action of the microprocessor complex, an alternative (non-conventional) pathway that generates miRNA precursors by splicing-mediated cleavage of short-hairpin introns (mirtrons) was discovered and characterized in invertebrates .
Following the processing of the hairpin-containing pre-miRNA into a linear dsRNA molecule, effector miRNA-containing ribonucleoprotein complexes (miRNPs) are assembled through a dynamic (and as yet not fully understood) process, that involves recruitment of one of the four argonaute (Ago) proteins , a glycine-tryptophan repeat-containing protein of 182 kDa (TNRC6, also known as GW182), as well as several other proteins (such as the CCR4-NOT deadenylase complex), which probably function as miRNP assembly or regulatory factors [16,17]. The functional role of the miRNP complex in the miRNA-guided RNA silencing pathway is to recognize the miRNA guide in the miRNA-miRNA* duplex, pair it with its target mRNA and prevent mRNA translation .
With very few exceptions, animal miRNAs regulate gene expression by imperfect base pairing with sequences on the 3'-UTR of target mRNAs and further miRNP-mediated inhibition of translation and/or mRNA destabilization [2,18]. Functional studies, as well as computational approaches, have shown that perfect or near-perfect complementarity between the mRNA and the nucleotides 2–8 on the 5'-region of the miRNA, also known as the “seed” region, is determinant for target mRNA recognition by the miRNA [19,20]. Insufficient 5'-pairing can nevertheless be partially compensated by strong base-pairing between the 3'-region of the miRNA and the target mRNA [19,21]. While the “seed” sequence of the mature miRNA dictates which mRNAs it potentially interacts with, it is the protein components of the miRNP complex (especially GW182) that direct and execute the silencing of target mRNAs .
The current knowledge indicates that miRNAs repress the initiation of translation by interfering with key components of the initiation step machinery, namely with 5' cap recognition and 40S small ribosomal subunit recruitment, thus hampering the association between the 60S ribosomal subunit and the 40S initiation complex, and thereby preventing the formation of an active ribosomal complex  or binding to PABP, the poly(A)-binding protein attached to the 3' end of the mRNA that is involved in the circularization of the mRNA . In addition to the repression of translation initiation, miRNAs can repress mRNA translation at the post-initiation steps by inhibiting ribosome elongation. Over the last few years, several studies surveying a large number of miRNA targets strongly indicate that mRNA destabilization (and consequent mRNA degradation) might nevertheless be a key factor in the decrease of protein levels caused by miRNAs. Unanimously, these studies found that miRNA repression results in concomitant changes in mRNA and protein levels, with changes in mRNA levels accounting for the majority, but not all, of the changes in protein abundance [25,26,27]. MiRNA-mediated mRNA decay is executed by the miRNP complexes through the recruitment of decay machinery components, leading to mRNA deadenylation and 5'-terminal decapping; the mRNA is subsequently degraded by the Xrn1 5'–3' exonuclease [28,29,30].
3. Role of MiRNAs in Cancer
The first indication that miRNA dysregulation could play a role in cancer was provided by Calin and colleagues, who demonstrated that two clustered miRNA genes, miR-15a and miR-16-1, were located in a region of the 13q14 locus that is commonly deleted in patients diagnosed with B-cell chronic lymphocytic leukemia (CLL) . Soon after, Croce and colleagues showed that approximately 50% of annotated human miRNAs are located in cancer-associated genomic regions (CAGRs), including fragile sites, minimal regions of loss of heterozygosity, minimal regions of amplification or common breakpoint regions . More recently, Kumar and colleagues demonstrated that global repression of miRNA maturation, through short hairpin (sh)RNA-mediated inhibition of several components of the miRNA processing machinery, promotes cellular transformation and tumorigenesis , thus supporting the idea that miRNAs play a crucial role in cancer progression. Indeed, several genome-wide miRNA-profiling studies provided evidence that distinct miRNA expression profiles distinguish tumors from normal tissues [34,35]. Different miRNA signatures were also associated with poor patient prognosis in lung cancer and CLL [36,37], indicating that miRNAs have potential to be used as diagnostic and prognostic markers.
Various genomic abnormalities were found to influence the activity of miRNAs, including deletions, amplifications or mutations involving miRNA loci, epigenetic silencing or dysregulation of transcription factors that target specific miRNAs. Loss or gain of miRNA function and its role in the development of cancer will be addressed below.
3.1. MiRNAs as Tumor Suppressors
MiRNAs can act as tumor suppressors when their reduced expression or loss of function contributes to the development of a malignant cell phenotype  (Figure 1). In this regard, accumulated data suggests that the cellular miRNA milieu is mostly composed of tumor suppressor miRNAs [33,34], and strong evidence has been provided for their role in cancer.
The loss of miR-15a and miR-16-1, due to chromosomal deletion of the locus 13q14 or germline mutation in their primary precursor, was associated with the development of the indolent form of CLL . Both miRNAs were found to regulate posttranscriptionally the expression of Bcl-2, an anti-apoptotic protein that is widely overexpressed in CLL , which supports the role of these miRNAs as tumor suppressors in CLL. Loss of miR-15a and miR-16-1 has also been observed in prostate cancer and multiple myeloma [40,41]. Similarly, members of the let-7 family of miRNAs were reported to map in genomic regions which are deleted in different human malignancies , and their downregulation is commonly observed in lung, breast and colon cancer [37,42,43]. The role of let-7 as tumor suppressor was clearly demonstrated in lung cancer, following the observation that downregulation of let-7 in lung tissues led to the constitutive overexpression of Ras and high-mobility group AT-hook 2 (HMGA2), both being oncoproteins that contribute to the pathogenesis of cancer [44,45,46]. Reduced let-7 expression was also shown to enhance c-Myc signaling in Burkitt lymphoma (BL) cells .
In addition to miR-15a/miR-16-1 and let-7, miR-29 family members (miR-29a, b, c) were shown to function as tumor suppressor miRNAs, their downregulation being associated with the development and progression of several human malignancies, including CLL, lung cancer, invasive breast cancer and hepatocellular carcinoma [36,37,43,48]. Interestingly, Fabbri and colleagues demonstrated that miR-29 can function as a tumor suppressor in lung cancer through interference with the methylation of tumor suppressor genes. By promoting the downregulation of the DNA methyltransferases 3A and B (DNMT3A and 3B), miR-29 induces re-expression of methylation-silenced tumor suppressor genes, such as the fragile histidine triad protein (FHIT) and the WW domain containing oxidoreductase (WWOX) .
The miR-34 family, which comprises miR-34a, b and c, has also received considerable attention for its potential role as tumor suppressor in several human malignancies. Decreased expression of miR-34 has been observed in lung, ovarian, CLL and colorectal cancer [50,51,52,53]. Reduced levels of miR-34a were also observed in human gliomas, which correlated with increased expression of the target oncogenes c-Met, Notch-1/2 and cyclin-dependent kinase 6 (CDK6) in glioma and stem cells . Interestingly, several reports indicate that miR-34 family members are direct transcriptional targets of the tumor suppressor p53 and suggest that some cellular roles of p53, including those related to the regulation of cellular proliferation and apoptosis, could be mediated by these miRNAs [55,56].
3.2. MiRNAs as Oncogenes
MiRNAs act as oncogenes when their increased expression or gain of function contributes to the development of a malignant cell phenotype  (Figure 1). One of the best-characterized oncogenic miRNAs is miR-17-92, a polycistronic cluster comprising six miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92-1), that maps at 13q31.3, a region amplified in several types of lymphoma and solid tumors [58,59]. Expression profiling studies revealed widespread overexpression of these miRNAs in a large number of human hematopoietic malignancies and solid tumors, including breast, colon, lung, pancreatic, prostate and stomach cancers [35,60]. The members of the miR-17-92 cluster were shown to promote tumor proliferation and induce angiogenesis through the activation of c-Myc [58,60,61], which is frequently activated in cancer. Interestingly, O’Donnell and colleagues also reported that the transcription of the miR-17-92 cluster is directly transactivated by c-Myc , which suggests the existence of a feedback mechanism to enhance pro-oncogenic signaling. The miR-17-92 cluster was also shown to enhance proliferation by activating several members of the E2F family of transcription factors—E2F1, E2F2, E2F3—which induce the expression of genes that drive cell cycle progression from G1 into S phase, and by inhibiting the cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21), a potent negative regulator of the G1-S checkpoint .
Strong evidence also suggests that miR-21 functions as an oncogene. Overexpression of this miRNA has been observed in numerous human malignancies, including colon, stomach, pancreas, prostate, lung, breast and liver cancer [35,43,64,65], being associated to important cancer hallmarks, such as uncontrolled cell proliferation, decreased apoptosis, invasion and migration . Several studies also demonstrated that miR-21 dysregulation contributes to the pathogenesis of glioblastoma (GBM) [67,68,69]. MiR-21 promotes tumor cell proliferation by inhibiting PDCD4 (programmed cell death protein 4), a tumor suppressor that prevents cell cycle progression via activation of the cyclin-dependent kinase 1 (Cdk1) inhibitor p21 . MiR-21 was also shown to target several components of p53, TGF-β and mitochondrial apoptotic networks, in order to decrease apoptotic activation in GBM cells , and to enhance tumor cell migration by inhibiting the matrix metalloproteinase (MMP) regulators RECK and TIMP3 .
Another miRNA with a clear role in the pathology of cancer is miR-10b. This miRNA is involved in the later stages of malignancy, by promoting the invasion of cancer cells into the surrounding stroma and metastasis to distant sites [73,74]. MiR-10b was found to be overexpressed in metastic samples of breast and hepatocellular carcinomas, when compared with tumor samples from metastasis-free patients [75,76], and patient samples from pancreatic adenocarcinomas  and glioblastomas , two types of extremely invasive/metastic cancer. It was proposed that miR-10b promotes invasion and metastasis by suppressing the translation of homeobox D10 (HOXD10), a transcriptional repressor known to inhibit the expression of several pro-metastatic genes, including the Ras homolog gene family member C (RHOC) . In addition to translation suppression of HOXD10, an E-cadherin-related mechanism has been proposed to explain the role of miR-10b in breast cancer metastasis .
MiRNA function can, nevertheless, vary according to the tissue and its transcriptome, including the miRNA targets expressed in that particular tissue . In addition to its well known oncogenic role, miR-17-5p, a member of the miR-17-92 cluster, was reported to be downregulated in breast cancer cells . Interestingly, decreased cellular proliferation was observed following the overexpression of miR-17-5p in cultured breast cancer cells, thus suggesting that this miRNA can act as a tumor suppressor in breast cancer . Similarly, the inhibition of miR-21 (a miRNA overexpressed in a wide variety of human tumors) was associated with increased cell growth in cervical cancer cells . Therefore, as stated by Croce and collaborators, miRNAs should not be classified as oncogenes or tumor suppressors unless the tissue or cell type involved in their action is specified .
Although the examples presented above constitute a small subset of miRNAs implicated in cancer development, they emphasize that targeting aberrantly expressed miRNAs has potential to impact future cancer therapies. Strategies for manipulating the expression of miRNAs will be discussed below.
4. Therapeutic Modulation of MiRNAs
Two major challenges are associated with the manipulation of miRNA function. The first concerns the identification of molecules that can effectively inhibit or “mimic” mature miRNAs, in order to achieve losses or gains of miRNA function, respectively. The second challenge concerns the efficient delivery of these molecules to the specific targeted sites.
4.1. Silencing of MiRNAs: Targeting MiRNAs Overexpressed in Cancer
The remarkable capacity of single-stranded or double-stranded DNA or RNA analogs to inhibit the activity of selected single-stranded genetic sequences has been explored in therapeutic approaches for several human gene-related diseases, including cancer. As mature miRNAs are short oligonucleotides, their inhibition can be achieved by base-pairing with complementary oligonucleotide sequences.
Multiple steps in the miRNA biogenesis pathway can be targeted with antisense oligonucleotides (ASOs) to repress miRNA production or function . In this regard, targeting the loop structure of the pre-miRNA was reported by Lee and colleagues as an interesting approach , although this was shown not to be very effective, possibly due to the difficulty in accessing the loop region. Inhibition of Drosha and Dicer processing of pri-miRNAs and pre-miRNAs, respectively, was achieved with morpholino ASOs in zebrafish . Although effective, this approach may have limited application in mammalian systems due to the slow turnover of the majority of mature mammalian miRNAs [84,85], which restrains the timing of inhibition of miRNA activity.
Currently, ASOs complementary to the mature miRNA (also known as anti-miRNA oligonucleotides, AMOs), and designed to block its function in the miRNP silencing complex, constitute the most effective technology for controlling miRNA expression, experimentally and/or therapeutically . In this regard, the addition of chemical groups to the 2'-hydroxyl group (at the C2 carbon of the ribose) was shown to be particularly effective in increasing the binding affinity for RNA and protecting the AMOs from nuclease degradation. Studies with AMOs containing methylated hydroxyl groups (2'-OMe) revealed increased resistance to nuclease cleavage and improved binding affinity to RNA compared to unmodified sequences [15,87]. When conjugated with a phosphorothioate backbone, intravenously-administered 2'-OMe-AMOs were also effective in inhibiting miRNA function in different animal tissues . The addition of methoxyethyl (2'-MOE) or fluorine (2'-F) groups further enhanced the activity of AMOs against the target miRNA, when compared to the simpler 2'-O-methyl modification . The strongest affinity for the target miRNA was, nevertheless, obtained with locked nucleic acid (LNA)-modified AMOs, which contain a methylene linker bridging the 2'-O-oxygen to the 4'-position that confers increased thermodynamic stability [90,91]. Although 2' modifications were shown to improve affinity to target RNA, their anti-miRNA activity was not fully correlated with affinity , suggesting that other variables may also be important for effective miRNA inhibition.
Synthetic polymers similar to RNA and DNA, designated peptide nucleic acids (PNAs), have also been described as excellent candidates for antisense therapies . As opposed to the ribose and deoxyribose sugar backbone, PNAs contain a polyamide backbone composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds . Since the backbone of PNA is neutrally charged (contains no charged phosphate groups), the binding between PNA and DNA or RNA strands is stronger than that between strands of DNA and/or RNA, where the electrostatic repulsions contribute to decrease the duplex stability . PNAs are not easily recognized by either nucleases or proteases, making them resistant to enzyme degradation, and can be easily modified to increase miRNA targeting . Indeed, unmodified PNAs cannot readily cross cell membranes to enter the cytosol and, therefore, PNAs are usually coupled to targeting molecules, such as CPPs, to improve cytosolic delivery . Efficient PNA-mediated miRNA inhibition was already reported in in vitro  and in vivo studies . In addition to the normal AMOs, which contain only one binding site for the target miRNA, a different class of miRNA inhibitors containing multiple binding sites per molecule-designated miRNA sponges—have been developed.
The concept of miRNA sponge was introduced by Ebert and coworkers . The authors reasoned that an mRNA-like transcript containing multiple complementary binding sites for an endogenous miRNA could bind the miRNA and block its activity. To achieve high levels of expression, they constructed plasmids encoding tandemly arrayed miRNA binding sites (MBS), driven by the CMV promoter. Aiming to prevent cleavage of the transcript containing the MBS, the authors introduced central mismatches in the miRNA/transcript duplex at positions 9–12. Upon transient transfection into mammalian cells and transcription by the RNA polymerase II, the transcripts (miRNA sponges) were at least as effective as LNA-modified AMOs in inhibiting not only one miRNA but also multiple members of a miRNA family . Based on the work of Ebert, Kluiver and colleagues developed a fast and versatile method to generate stably-expressed miRNA sponges containing 10 or more miRNA binding sites . Moreover, the authors reported that constructions containing multiple binding sites for two different miRNAs were efficient in inhibiting simultaneously both target miRNAs.
Since overexpression of miRNAs has been associated with several steps of the tumorigenic process, modulating their levels could provide therapeutic benefit. Indeed, encouraging results from in vitro and in vivo studies using AMO-based strategies have already been achieved.
Due to its considerable overexpression in a wide range of human tumors, miR-21 has been targeted in several anti-cancer AMO-based strategies. Knockdown of miR-21 in cultured hepatocellular cancer cells resulted in increased apoptosis and suppressed cell growth , while AMO-mediated miR-21 inhibition in androgen-independent prostate cancer cell lines (DU145 and PC-3) increased cell sensitivity to apoptosis and inhibited cell motility and invasion, without affecting tumor cell proliferation . Similarly, transfection of breast cancer cells with anti-miR-21 oligonucleotides suppressed both cell proliferation in vitro and tumor growth in a xenograft mouse model . Seike and colleagues reported that EGFR mutations are generally associated with increased miR-21 expression in nonsmoking lung cancer patients, and AMO-mediated miR-21 knockdown sensitized cancer cells to the EGFR-tyrosine kinase inhibitor AG1478 . In a separate study, transgenic manipulation of miR-21 and the targeted delivery of anti-miR-21 oligonucleotides were shown to slow down tumor progression in a Ras-driven murine model of lung cancer .
Two different studies in cultured pancreatic cancer cells demonstrated that oligonucleotide-mediated miR-221 silencing results in increased apoptotic activity, decreased tumor cell proliferation  and increased cytotoxicity of the anti-cancer agent benzyl isothiocyanate . Furthermore, 2'-O-Me phosphorothioate-modified anti-miR-221 oligonucleotides were shown to decrease proliferation of cultured hepatocellular cancer cells . When tested in a mouse model of disease, the administration of a cholesterol-modified isoform of anti-miR-221 not only improved pharmacokinetics and liver tissue distribution, compared to unmodified oligonucleotide, but also reduced miR-221 levels in the liver (within a week of intravenous administration), produced significant antitumor activity and increased animal survival . Similarly, the therapeutic silencing of miR-10b with cholesterol-modified AMOs (antagomirs) suppressed metastasis in a mouse mammary tumor model .
MiRNA sponges were also shown to be effective in inhibiting miRNA activity. Kluiver and colleagues demonstrated that the combined inhibition of miRNAs of the miR-17-92 cluster, using sponges containing binding sites for several elements of this family, was significantly more effective in inhibiting the proliferation of cultured B-cell lymphoma cells, than individual miRNAs .
4.2. Overexpression of MiRNAs: Re-Expressing MiRNAs Downregulated in Cancer
Two different strategies, involving miRNA mimics and plasmid or virally-encoded miRNA constructs, have been widely used to restore miRNA expression in cells, aiming at a therapeutic benefit.
MiRNA mimics are double-stranded RNA molecules similar to the endogenous Dicer product (miRNA:miRNA* duplex), composed of a guide strand identical to the mature miRNA and a passenger strand that is partially or fully complementary to the guide strand . Due to their unfavorable physicochemical characteristics for in vivo administration, miRNA mimics can be chemically modified to increase protection from nuclease degradation, decrease innate immune system activation, reduce the incidence of off-target effects and improve pharmacodynamics . In this regard, the addition of methyl (2'-O-Me), methoxyethyl (2'-MOE) or fluorine (2'-F) groups (or their combination) to the ribose ring was shown to enhance the stability of miRNA mimics [109,110]. Aromatic compounds, such as 3'-benzene-pyridine, were able to increase protection from nuclease degradation and enhance activity when added to the 3' end of miRNA mimics . The guided strand can also be modified to enhance the miRNA activity in the miRNP complex. Indeed, the addition of 2'-OMe modifications at the 3' end and the presence of a 2-nucleotide (nt) 3' overhang assist with miRNP loading and degradation of the passenger strand [107,112].
Recently, a new class of miRNA mimics has been described. As opposed to the traditional double-stranded miRNA mimics, Chorn and colleagues demonstrated that modified single-stranded miRNA mimics also exhibit significant argonaute-mediated miRNA seed-based activity in cultured cells . However, the potency of single-stranded mimics was inferior to that achieved with double-stranded oligonucleotides, which suggested that additional modifications should be explored, aiming at improving the efficacy of these molecules .
An alternative strategy for therapeutic miRNA replacement involves the expression of a shRNA or pri-miRNA mimic from a plasmid or viral construct. When compared to the delivery of double-stranded miRNA mimics, this approach provides a more stable expression of the mature miRNA and allows the expression of multiple miRNAs from one transcript .
ShRNAs are structurally similar to pre-miRNAs, with a base-paired stem, a small loop and a 3'-end UU overhang . Expression of shRNAs is usually driven by RNA polymerase III promoters, including the H1 and U6, as they are involved in the production of small cellular transcripts and use precise initiation and termination sites [115,116]. However, both promote high levels of shRNA expression, which can elicit toxicity . Indeed, overexpression of shRNAs was already shown to saturate both exportin-5 and Ago2, which triggers a global dysfunction of the endogenous miRNA pathway [115,118]. As opposed to shRNAs, pri-miRNA mimics are usually transcribed from low expression RNA polymerase II promoters, which can drive tissue-specific expression , and further processed by Drosha, thus reducing the risk of saturation of the endogenous miRNA pathway. In this regard, reduced in vivo toxicity was reported following miRNA re-expression via pri-miRNA mimics, when compared to that observed following miRNA re-expression via shRNAs [119,120]. Pri-miRNA mimics were, nevertheless, suggested to be less efficient in promoting the silencing of target mRNAs, when compared to matched shRNAs [119,120].
A common hurdle to both shRNAs and pri-miRNA mimics is the existence of off-target effects associated with their application. As stated by Sybley and collaborators, complementary base pairing with as little as 8-nt homology is sufficient to cause translational repression of nontarget genes when multiple target matches are present within their 3' UTRs [117,121]. Therefore, careful design of shRNA or pri-miRNA mimics constructs should be considered in order to reduce the off-target effects associated with these technologies.
Encouraging results from in vitro and in vivo studies using miRNA-expressing strategies have also reported. Enforced expression of miR-34a in human cultured p53-mutant prostate cancer (PCa) cells induced cell-cycle arrest, apoptosis or senescence  and, most importantly, re-expression of this miRNA in CD44+ PCa cells blocked tumor progression and metastasis following orthotopic tumor cell implantation in immunocompromised mice . Similarly, re-expression of miR-34a in human colon cancer cells (HCT116 and RKO) caused a significant inhibition of cell proliferation and induced senescence-like phenotypes in these cell lines . Importantly, intratumoral administration of complexes prepared by association of miR-34a and atelocollagen suppressed tumor growth in subcutaneous mice xenograft tumors . In a different study, reduced tumor cell proliferation, migration and invasion were observed in cultured breast cancer cells following exogenous expression of miR-34a .
The transient overexpression of let-7a and let-7f, two dominant isoforms of the let-7 family in A549 lung adenocarcinoma cells, suppressed tumor cell proliferation . Similarly, overexpression of let-7 in these cells and HepG2 liver cancer cells repressed cell cycle progression and cell division . Let-7 overexpression was also reported to induce apoptosis and cell cycle arrest in colon cancer and Burkitt's lymphoma (BL) cell lines [41,46].
Decreased expression of members of the miR-29 family (miR-29a, b, c) was observed in various malignancies that contain aberrant DNA hypermethylation patterns, including lung cancer [48,127]. In this regard, the enforced expression of miR-29 in lung cancer cell lines led to reduced global DNA methylation, induced re-expression of methylation-silenced tumor suppressor genes, such as FHIT and WWOX, and inhibited tumor cell proliferation in vitro and tumor growth in vivo . In CLL, the loss of miR-15a and miR-16-1 was associated with decreased apoptotic activity due to the overexpression of the anti-apoptotic protein Bcl-2, while miR-15a/miR-16-1 reconstitution increased apoptosis through repression of Bcl-2 mRNA translation . Furthermore, overexpression of miR-7, a miRNA involved in the repression of the pro-oncogenic Akt pathway, reduced the proliferation and invasion in different GBM cell lines and in one GBM stem cell line , while expression of miR-128 inhibited the proliferation of glioma cells by decreasing the levels of E2F3a . Figure 2 depicts the different strategies developed for the therapeutic modulation of miRNA expression.
Although modified AMOs and miRNA mimics have been successfully tested for miRNA modulation in liver diseases [130,131,132], including hepatocellular cancer [105,133], by taking advantage of the physiological role of the liver as the major blood filtration organ. Nevertheless, their widespread application to other non-hepatic diseases is restricted by the lack of tissue specificity. Being negatively charged molecules, miRNA inhibitors and miRNA mimics do not readily bind to cell plasma membrane and, with very few exceptions , do not cross the blood-brain barrier [135,136], which limits their use to the treatment of brain malignancies. Therefore, the successful in vivo nucleic acid delivery requires the development of carriers that can increase bioavailability, protect the nucleic acids from nuclease degradation and enhance their uptake by the target cells, while sparing the normal tissues.
4.3. Delivery of Nucleic Acids to Modulate miRNA Function
The discovery of RNAi-mediated mechanisms of gene silencing and the development of suitable carriers for in vitro and in vivo delivery of siRNAs and shRNAs [118,137], paved the way for establishing technical approaches that can be applied in miRNA-based therapies. Viral and non-viral vectors, particularly lipid- and polymer-based carriers, have been successfully tested for siRNA/shRNA delivery in preclinical studies [138,139], and, more recently, employed for delivery of AMOs or miRNA-expressing constructs in both cellular and animal models.
4.3.1. Viral Vectors
Due to their high transduction efficiency, viral vectors have been extensively used for delivering miRNA-expressing constructs to cancer cells in miRNA-based therapeutics (Table 1). The systemic administration of an AAV8 encoding miR-26a, which is highly downregulated in hepatocellular cancer and induces cell cycle arrest by targeting the cyclins D2 and E2, resulted in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis and significant tumor regression, without toxicity . Similarly, the intranasal administration of adenovirally-coded let-7 significantly reduced tumor burden in an orthotopic mouse model of NSCLC . Lentiviral vectors were also successfully used for the reestablishment of miRNA expression in tumors. In a xenograft prostate cancer mice model, the intratumoral injection of lentivirally-coded miR-15-16 led to growth arrest within 1 week of treatment and considerable volume regression thereafter, whereas no significant alterations in tumor growth were observed in animals treated with an empty viral vector . Similarly, the intratumoral administration of lentiviruses coding for a sequence targeting miR-21 was shown to enhance tumor cell death by apoptosis and, when combined with the anti-neoplastic drug gentabicine, to induce tumor regression .
|Table 1. MiRNAs as targets for cancer therapy through delivery of anti-miRNA oligonucleotides or miRNA-expressing constructs mediated by viral and non-viral vectors.|
|Carrier||Disease||Target miRNA (s) and
role in cancer
|Viruses||Adeno-associated viruses(AAV8)||Hepatocellular |
|miR-26 tumor suppressor|||
|Adenoviruses||Lung cancer||let-7 tumor suppressor|||
|Adenoviruses||Glioblastoma||miR-145 tumor suppressor|||
|Lentiviruses||Prostate cancer||miR-15-16 tumor suppressor|||
|Lentiviruses||Pancreatic cancer||miR-21 oncogene|||
|Lipid-based nanoparticles||Cationic liposomes||Breast cancer||miR-34a tumor suppressor|||
|Cationic liposomes||Pancreatic cancer||miR-34a, miR-143-145 tumor suppressors|||
|Neutral lipid emulsion©||Lung cancer||miR-34a, let-7 tumor suppressors|||
|Stable nucleic acid lipid particles||Glioblastoma||miR-21 oncogene|||
|Polymer-based nanoparticles||Polyurethane||Glioblastoma||miR-145 tumor suppressor|||
|Poly(lactic-co-glycolic acid)||Lymphoma||miR-155 oncogene|||
Viral vectors have also been applied in miRNA-based therapeutic strategies towards GBM. Lee and colleagues took advantage of the abundant expression of the enzyme telomerase reverse transcriptase (hTERT) in cancer cells and developed a multimodal GBM-targeting approach, combining hTERT-targeting ribozyme-controlled HSV-tk expression with overexpression of miR-145, a miRNA that is usually downregulated in GBM . For this purpose, the authors constructed adenoviral vectors that express hTERT.Rz.HSVtk and miR-145 under control of the CMV promoter, to ensure high expression of the transgene in the target cells. In a xenograft mice model, the intratumoral administration of the adenovirus harboring the HSV-tk expression cassette plus miR-145, combined with intraperitoneal injection of ganciclovir, resulted in increased animal survival, when compared to that observed with the administration of virus coding for HSV-tk or miR-145 per se . Adenoviruses encoding shRNAs targeting mature miRNAs have also been developed for GBM therapy. Wang and colleagues constructed an adenoviral vector expressing shRNAs that co-repress the expression of miR-221 and miR-222 . Upon transduction of cultured GBM cells, decreased levels of these miRNAs were detected, which were associated with increased expression of their target p27kip1, cell cycle arrest in G1 phase and increased apoptosis .
4.3.2. Non-Viral Vectors
Synthetic non-viral vectors, including lipid- and polymer-based nanoparticles, have also been widely tested for the delivery of miRNA-based nucleic acids into tumor cells, as illustrated in Table 1.
Cationic liposome-mediated delivery of a plasmid encoding miR-34a (T-VISA-miR-34a) to cultured breast cancer cells was shown to result in the downregulation of several miR-34a target genes leading to significant suppression of breast cancer cell growth, migration and invasion . Furthermore, intravenous injection of T-VISA-miR-34a: liposomal complex nanoparticles significantly inhibited tumor growth, prolonged survival, and did not induce systemic toxicity in an orthotopic mouse model of breast cancer . Similarly, Pramanik and colleagues demonstrated that systemic intravenous delivery of liposome-formulated plasmid-encoded miR-34a or miR-143-145 inhibited tumor growth in subcutaneous and orthotopic pancreas MiaPaca-2 xenografts . A different lipid-based formulation was also employed for the delivery of miR-34a and let-7 to a Kras-driven NSCLC mouse model. Trang and colleagues used a neutral lipid emulsion (NLE) that, when combined with synthetic miRNA mimics, forms nanoparticles in the nanometer diameter range and with a surface net charge close to zero . In this regard, following intravenous administration of the lipid-based nanoparticles, a significant reduction in tumor growth was observed for animals treated with nanoparticles containg miR-34a or let-7, when compared to that observed for animals treated with nanoparticles containing a control miRNA mimic . Importantly, a liposome-formulated miR-34a mimic is currently being tested in a phase I clinical trial in patients with unresectable primary liver cancer or metastatic cancer with liver involvement . We have recently developed stable nucleic acid lipid particles (SNALPs), which were targeted towards GBM cells by covalent coupling of the peptide chlorotoxin (CTX) to the liposomal surface . Our studies demonstrated that CTX-coupled SNALPs are able to efficiently and specifically deliver encapsulated anti-miR-21 oligonucleotides to cultured U87 GBM cells (Figure 3a), which resulted in a significant decrease in miR-21 expression, increased levels of the tumor suppressors PTEN and PDCD4, caspase activation (Figure 3b) and increased cytotoxicity of the tyrosine kinase inhibitor sunitinib. Importantly, in vivo studies revealed that the attachment of CTX to the liposomal surface enhances SNALP internalization into established intracranial tumors, when compared to that obtained for the non-targeted SNALPs (Figure 3c).
Delivery of miRNA-based nucleic acids, namely AMOs and miRNA mimics, to tumor cells has also been accomplished by using polymer-based nanoparticles. Some of the most commonly employed polymers include polyurethanes, poly(lactic-co-glycolic acid) (PLGA) and polyamidoamine (PAMAM) dendrimers (Table 1).
Polyurethanes are conventionally used in tissue engineering and gene delivery due to their biocompatibility and physicochemical properties [149,152]. When combined with PEI, cationic polyurethane (PU)-shortbranch PEI (PU-PEI) was shown to exhibit high transfection efficiency and low cytotoxicity in vitro and in vivo [149,153]. Using PU-PEI as a delivery vehicle, Yang and colleagues reported efficient delivery of miR-145 to CD133+ GBM cells, which resulted in a significant decrease in their tumorigenic potential and facilitated differentiation into CD133-negative cells . Moreover, PU-PEI-mediated miR-145 expression in CD133+ cells suppressed the expression of anti-apoptotic and drug-resistance genes, while increasing the cell sensitivity to radiation and temozolomide (TMZ). When administered intratumorally in an orthotopic GBM-CD133+ xenograft mouse model, nanoparticle-formulated miR145 significantly reduced tumorigenesis and improved animal survival when combined with radiotherapy and TMZ, compared to that observed in animals treated with PU-PEI per se .
PLGA-based nanoparticles have also been extensively used to enhance the delivery of therapeutic agents to target cells, due to their biocompatibility and biodegradability . In this regard, Babar and colleagues developed a delivery system by combining PLGA with PNAs targeting the oncogenic miR-155, and attached the CPP penetratin to the surface of the PLGA nanoparticles, in order to enhance cellular uptake . Following incubation of cultured lymphoma cells with the generated nanoparticles (ANTP-NP), a significant increase in particle internalization, decreased expression of miR-155 and increased levels of the miR-155 target SHIP1 were achieved, when compared to that observed for cells incubated with nanoparticles lacking penetratin or nanoparticles loaded with a scrambled control. Moreover, upon intratumoral or systemic administration of ANTP-NP, a moderate (although significant) delay in tumor growth was detected in a subcutaneous xenograft mouse model of lymphoma .
Due to the presence of positively charged amino groups on their surface, PAMAM dendrimers can easily interact with nucleic acids to form complexes through charge-based interactions and protect them from nuclease degradation, while exhibiting minimal toxicity [150,155]. Moreover, the open nature of the dendritic architecture enables the entrapment of drugs within their core through electrostatic, hydrophobic and hydrogen bond interactions . Ren and colleagues used PAMAM dendrimers as a carrier to co-deliver anti-miR-21 oligonucleotides and the drug 5-FU to human GBM cells and reported a significant increase of apoptosis and enhanced cytotoxicity of 5-FU, associated with a decrease in the invasive capacity of U251 GBM cells .
MiRNAs are a class of subtle gene regulators that, by targeting oncogenes and tumor suppressors, have the ability to modulate key cellular processes that define the cell phenotype. Abnormal expression of these small molecules was shown to play a causal role in different steps of tumorigenesis, from initiation and development to metastatic progression. Importantly, miRNA-based therapeutic strategies have been successfully applied in pre-clinical models for several human malignancies, thus emphasizing the enormous potential of miRNAs as therapeutic targets for neoplastic diseases. Future clinical trials should provide new insights into the safety and efficacy of the developed miRNA-based anti-cancer therapies.
This work was supported by the Portuguese Foundation for Science and Technology and FEDER/COMPETE (grants PTDC/DTP-FTO/0265/2012 and PEst-C/SAU/LA0001/20119). P.M.C. is recipient of a fellowship from the Portuguese Foundation for Science and Technology (SFRH/BD/45902/2008).
Conflicts of Interest
The authors declare no conflict of interest.
- Alvarez-Garcia, I.; Miska, E.A. Microrna functions in animal development and human disease. Development 2005, 132, 4653–4662, doi:10.1242/dev.02073.
- Ambros, V. The functions of animal micrornas. Nature 2004, 431, 350–355, doi:10.1038/nature02871.
- Croce, C.M. Causes and consequences of microrna dysregulation in cancer. Nat. Rev. Genet. 2009, 10, 704–714, doi:10.1038/nrg2634.
- Stark, A.; Brennecke, J.; Bushati, N.; Russell, R.B.; Cohen, S.M. Animal micrornas confer robustness to gene expression and have a significant impact on 3'utr evolution. Cell 2005, 123, 1133–1146, doi:10.1016/j.cell.2005.11.023.
- Liu, N.; Olson, E.N. Microrna regulatory networks in cardiovascular development. Dev. Cell 2010, 18, 510–525, doi:10.1016/j.devcel.2010.03.010.
- Gangaraju, V.K.; Lin, H. Micrornas: Key regulators of stem cells. Nat. Rev. Mol. Cell Biol. 2009, 10, 116–125, doi:10.1038/nrm2621.
- O'Connell, R.M.; Rao, D.S.; Chaudhuri, A.A.; Baltimore, D. Physiological and pathological roles for micrornas in the immune system. Nat. Rev. Immunol. 2010, 10, 111–122, doi:10.1038/nri2708.
- Poy, M.N.; Eliasson, L.; Krutzfeldt, J.; Kuwajima, S.; Ma, X.; Macdonald, P.E.; Pfeffer, S.; Tuschl, T.; Rajewsky, N.; Rorsman, P.; et al. A pancreatic islet-specific microrna regulates insulin secretion. Nature 2004, 432, 226–230, doi:10.1038/nature03076.
- Lecellier, C.H.; Dunoyer, P.; Arar, K.; Lehmann-Che, J.; Eyquem, S.; Himber, C.; Saib, A.; Voinnet, O. A cellular microrna mediates antiviral defense in human cells. Science 2005, 308, 557–560, doi:10.1126/science.1108784.
- Nelson, P.T.; Wang, W.X.; Rajeev, B.W. Micrornas (mirnas) in neurodegenerative diseases. Brain Pathol. 2008, 18, 130–138, doi:10.1111/j.1750-3639.2007.00120.x.
- Small, E.M.; Olson, E.N. Pervasive roles of micrornas in cardiovascular biology. Nature 2011, 469, 336–342, doi:10.1038/nature09783.
- Pandey, A.K.; Agarwal, P.; Kaur, K.; Datta, M. Micrornas in diabetes: Tiny players in big disease. Cell. Physiol. Biochem. 2009, 23, 221–232, doi:10.1159/000218169.
- Calin, G.A.; Croce, C.M. Microrna signatures in human cancers. Nat. Rev. Cancer 2006, 6, 857–866, doi:10.1038/nrc1997.
- Ruby, J.G.; Jan, C.H.; Bartel, D.P. Intronic microrna precursors that bypass drosha processing. Nature 2007, 448, 83–86, doi:10.1038/nature05983.
- Meister, G.; Landthaler, M.; Patkaniowska, A.; Dorsett, Y.; Teng, G.; Tuschl, T. Human argonaute2 mediates rna cleavage targeted by mirnas and sirnas. Mol. Cell 2004, 15, 185–197, doi:10.1016/j.molcel.2004.07.007.
- Filipowicz, W.; Bhattacharyya, S.N.; Sonenberg, N. Mechanisms of post-transcriptional regulation by micrornas: Are the answers in sight? Nat. Rev. Genet. 2008, 9, 102–114.
- Perron, M.P.; Provost, P. Protein interactions and complexes in human microrna biogenesis and function. Front. Biosci. 2008, 13, 2537–2547, doi:10.2741/2865.
- Bartel, D.P. Micrornas: Genomics, biogenesis, mechanism, and function. Cell 2004, 116, 281–297, doi:10.1016/S0092-8674(04)00045-5.
- Brennecke, J.; Stark, A.; Russell, R.B.; Cohen, S.M. Principles of microrna-target recognition. PLoS Biol. 2005, 3, e85, doi:10.1371/journal.pbio.0030085.
- Liu, X.; Fortin, K.; Mourelatos, Z. Micrornas: Biogenesis and molecular functions. Brain Pathol. 2008, 18, 113–121, doi:10.1111/j.1750-3639.2007.00121.x.
- Kiriakidou, M.; Nelson, P.T.; Kouranov, A.; Fitziev, P.; Bouyioukos, C.; Mourelatos, Z.; Hatzigeorgiou, A. A combined computational-experimental approach predicts human microrna targets. Genes Dev. 2004, 18, 1165–1178, doi:10.1101/gad.1184704.
- Fabian, M.R.; Sonenberg, N. The mechanics of mirna-mediated gene silencing: A look under the hood of mirisc. Nat. Struct. Mol. Biol. 2012, 19, 586–593, doi:10.1038/nsmb.2296.
- Chendrimada, T.P.; Finn, K.J.; Ji, X.; Baillat, D.; Gregory, R.I.; Liebhaber, S.A.; Pasquinelli, A.E.; Shiekhattar, R. Microrna silencing through risc recruitment of eif6. Nature 2007, 447, 823–828, doi:10.1038/nature05841.
- Fabian, M.R.; Mathonnet, G.; Sundermeier, T.; Mathys, H.; Zipprich, J.T.; Svitkin, Y.V.; Rivas, F.; Jinek, M.; Wohlschlegel, J.; Doudna, J.A.; et al. Mammalian mirna risc recruits caf1 and pabp to affect pabp-dependent deadenylation. Mol. Cell 2009, 35, 868–880, doi:10.1016/j.molcel.2009.08.004.
- Baek, D.; Villen, J.; Shin, C.; Camargo, F.D.; Gygi, S.P.; Bartel, D.P. The impact of micrornas on protein output. Nature 2008, 455, 64–71, doi:10.1038/nature07242.
- Guo, H.; Ingolia, N.T.; Weissman, J.S.; Bartel, D.P. Mammalian micrornas predominantly act to decrease target mrna levels. Nature 2010, 466, 835–840, doi:10.1038/nature09267.
- Selbach, M.; Schwanhausser, B.; Thierfelder, N.; Fang, Z.; Khanin, R.; Rajewsky, N. Widespread changes in protein synthesis induced by micrornas. Nature 2008, 455, 58–63, doi:10.1038/nature07228.
- Behm-Ansmant, I.; Rehwinkel, J.; Doerks, T.; Stark, A.; Bork, P.; Izaurralde, E. Mrna degradation by mirnas and gw182 requires both ccr4:Not deadenylase and dcp1:Dcp2 decapping complexes. Genes Dev. 2006, 20, 1885–1898, doi:10.1101/gad.1424106.
- Giraldez, A.J.; Mishima, Y.; Rihel, J.; Grocock, R.J.; Van Dongen, S.; Inoue, K.; Enright, A.J.; Schier, A.F. Zebrafish mir-430 promotes deadenylation and clearance of maternal mrnas. Science 2006, 312, 75–79, doi:10.1126/science.1122689.
- Wu, L.; Fan, J.; Belasco, J.G. Micrornas direct rapid deadenylation of mrna. Proc. Natl. Acad. Sci. USA 2006, 103, 4034–4039, doi:10.1073/pnas.0510928103.
- Calin, G.A.; Dumitru, C.D.; Shimizu, M.; Bichi, R.; Zupo, S.; Noch, E.; Aldler, H.; Rattan, S.; Keating, M.; Rai, K.; et al. Frequent deletions and down-regulation of micro-rna genes mir15 and mir16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 2002, 99, 15524–15529, doi:10.1073/pnas.242606799.
- Calin, G.A.; Sevignani, C.; Dumitru, C.D.; Hyslop, T.; Noch, E.; Yendamuri, S.; Shimizu, M.; Rattan, S.; Bullrich, F.; Negrini, M.; et al. Human microrna genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci. USA 2004, 101, 2999–3004, doi:10.1073/pnas.0307323101.
- Kumar, M.S.; Lu, J.; Mercer, K.L.; Golub, T.R.; Jacks, T. Impaired microrna processing enhances cellular transformation and tumorigenesis. Nat. Genet. 2007, 39, 673–677, doi:10.1038/ng2003.
- Lu, J.; Getz, G.; Miska, E.A.; Alvarez-Saavedra, E.; Lamb, J.; Peck, D.; Sweet-Cordero, A.; Ebert, B.L.; Mak, R.H.; Ferrando, A.A.; et al. Microrna expression profiles classify human cancers. Nature 2005, 435, 834–838, doi:10.1038/nature03702.
- Volinia, S.; Calin, G.A.; Liu, C.G.; Ambs, S.; Cimmino, A.; Petrocca, F.; Visone, R.; Iorio, M.; Roldo, C.; Ferracin, M.; et al. A microrna expression signature of human solid tumors defines cancer gene targets. Proc. Natl. Acad. Sci. USA 2006, 103, 2257–2261, doi:10.1073/pnas.0510565103.
- Calin, G.A.; Ferracin, M.; Cimmino, A.; Di Leva, G.; Shimizu, M.; Wojcik, S.E.; Iorio, M.V.; Visone, R.; Sever, N.I.; Fabbri, M.; et al. A microrna signature associated with prognosis and progression in chronic lymphocytic leukemia. N. Engl. J. Med. 2005, 353, 1793–1801, doi:10.1056/NEJMoa050995.
- Yanaihara, N.; Caplen, N.; Bowman, E.; Seike, M.; Kumamoto, K.; Yi, M.; Stephens, R.M.; Okamoto, A.; Yokota, J.; Tanaka, T.; et al. Unique microrna molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 2006, 9, 189–198, doi:10.1016/j.ccr.2006.01.025.
- Esquela-Kerscher, A.; Slack, F.J. Oncomirs—Micrornas with a role in cancer. Nat. Rev. Cancer 2006, 6, 259–269, doi:10.1038/nrc1840.
- Cimmino, A.; Calin, G.A.; Fabbri, M.; Iorio, M.V.; Ferracin, M.; Shimizu, M.; Wojcik, S.E.; Aqeilan, R.I.; Zupo, S.; Dono, M.; et al. Mir-15 and mir-16 induce apoptosis by targeting bcl2. Proc. Natl. Acad. Sci. USA 2005, 102, 13944–13949, doi:10.1073/pnas.0506654102.
- Ambs, S.; Prueitt, R.L.; Yi, M.; Hudson, R.S.; Howe, T.M.; Petrocca, F.; Wallace, T.A.; Liu, C.G.; Volinia, S.; Calin, G.A.; et al. Genomic profiling of microrna and messenger rna reveals deregulated microrna expression in prostate cancer. Cancer Res. 2008, 68, 6162–6170, doi:10.1158/0008-5472.CAN-08-0144.
- Roccaro, A.M.; Sacco, A.; Thompson, B.; Leleu, X.; Azab, A.K.; Azab, F.; Runnels, J.; Jia, X.; Ngo, H.T.; Melhem, M.R.; et al. Micrornas 15a and 16 regulate tumor proliferation in multiple myeloma. Blood 2009, 113, 6669–6680, doi:10.1182/blood-2009-01-198408.
- Akao, Y.; Nakagawa, Y.; Naoe, T. Let-7 microrna functions as a potential growth suppressor in human colon cancer cells. Biol. Pharm. Bull. 2006, 29, 903–906, doi:10.1248/bpb.29.903.
- Iorio, M.V.; Ferracin, M.; Liu, C.G.; Veronese, A.; Spizzo, R.; Sabbioni, S.; Magri, E.; Pedriali, M.; Fabbri, M.; Campiglio, M.; et al. Microrna gene expression deregulation in human breast cancer. Cancer Res. 2005, 65, 7065–7070, doi:10.1158/0008-5472.CAN-05-1783.
- Esquela-Kerscher, A.; Trang, P.; Wiggins, J.F.; Patrawala, L.; Cheng, A.; Ford, L.; Weidhaas, J.B.; Brown, D.; Bader, A.G.; Slack, F.J. The let-7 microrna reduces tumor growth in mouse models of lung cancer. Cell Cycle 2008, 7, 759–764, doi:10.4161/cc.7.6.5834.
- Johnson, S.M.; Grosshans, H.; Shingara, J.; Byrom, M.; Jarvis, R.; Cheng, A.; Labourier, E.; Reinert, K.L.; Brown, D.; Slack, F.J. Ras is regulated by the let-7 microrna family. Cell 2005, 120, 635–647, doi:10.1016/j.cell.2005.01.014.
- Lee, Y.S.; Dutta, A. The tumor suppressor microrna let-7 represses the hmga2 oncogene. Genes Dev. 2007, 21, 1025–1030, doi:10.1101/gad.1540407.
- Sampson, V.B.; Rong, N.H.; Han, J.; Yang, Q.; Aris, V.; Soteropoulos, P.; Petrelli, N.J.; Dunn, S.P.; Krueger, L.J. Microrna let-7a down-regulates myc and reverts myc-induced growth in burkitt lymphoma cells. Cancer Res. 2007, 67, 9762–9770, doi:10.1158/0008-5472.CAN-07-2462.
- Xiong, Y.; Fang, J.H.; Yun, J.P.; Yang, J.; Zhang, Y.; Jia, W.H.; Zhuang, S.M. Effects of microrna-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma. Hepatology 2010, 51, 836–845.
- Fabbri, M.; Garzon, R.; Cimmino, A.; Liu, Z.; Zanesi, N.; Callegari, E.; Liu, S.; Alder, H.; Costinean, S.; Fernandez-Cymering, C.; et al. Microrna-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3a and 3b. Proc. Natl. Acad. Sci. USA 2007, 104, 15805–15810, doi:10.1073/pnas.0707628104.
- Corney, D.C.; Hwang, C.I.; Matoso, A.; Vogt, M.; Flesken-Nikitin, A.; Godwin, A.K.; Kamat, A.A.; Sood, A.K.; Ellenson, L.H.; Hermeking, H.; et al. Frequent downregulation of mir-34 family in human ovarian cancers. Clin. Cancer Res. 2010, 16, 1119–1128, doi:10.1158/1078-0432.CCR-09-2642.
- Fabbri, M.; Bottoni, A.; Shimizu, M.; Spizzo, R.; Nicoloso, M.S.; Rossi, S.; Barbarotto, E.; Cimmino, A.; Adair, B.; Wojcik, S.E.; et al. Association of a microrna/tp53 feedback circuitry with pathogenesis and outcome of b-cell chronic lymphocytic leukemia. JAMA 2011, 305, 59–67, doi:10.1001/jama.2010.1919.
- Tanaka, N.; Toyooka, S.; Soh, J.; Kubo, T.; Yamamoto, H.; Maki, Y.; Muraoka, T.; Shien, K.; Furukawa, M.; Ueno, T.; et al. Frequent methylation and oncogenic role of microrna-34b/c in small-cell lung cancer. Lung Cancer 2011, 76, 32–38.
- Vogt, M.; Munding, J.; Gruner, M.; Liffers, S.T.; Verdoodt, B.; Hauk, J.; Steinstraesser, L.; Tannapfel, A.; Hermeking, H. Frequent concomitant inactivation of mir-34a and mir-34b/c by cpg methylation in colorectal, pancreatic, mammary, ovarian, urothelial, and renal cell carcinomas and soft tissue sarcomas. Virchows Arch. 2011, 458, 313–322, doi:10.1007/s00428-010-1030-5.
- Li, Y.; Guessous, F.; Zhang, Y.; Dipierro, C.; Kefas, B.; Johnson, E.; Marcinkiewicz, L.; Jiang, J.; Yang, Y.; Schmittgen, T.D.; et al. Microrna-34a inhibits glioblastoma growth by targeting multiple oncogenes. Cancer Res. 2009, 69, 7569–7576, doi:10.1158/0008-5472.CAN-09-0529.
- He, L.; He, X.; Lim, L.P.; de Stanchina, E.; Xuan, Z.; Liang, Y.; Xue, W.; Zender, L.; Magnus, J.; Ridzon, D.; et al. A microrna component of the p53 tumour suppressor network. Nature 2007, 447, 1130–1134, doi:10.1038/nature05939.
- Rokhlin, O.W.; Scheinker, V.S.; Taghiyev, A.F.; Bumcrot, D.; Glover, R.A.; Cohen, M.B. Microrna-34 mediates ar-dependent p53-induced apoptosis in prostate cancer. Cancer Biol. Ther. 2008, 7, 1288–1296, doi:10.4161/cbt.7.8.6284.
- Garzon, R.; Calin, G.A.; Croce, C.M. Micrornas in cancer. Annu. Rev. Med. 2009, 60, 167–179, doi:10.1146/annurev.med.59.053006.104707.
- He, L.; Thomson, J.M.; Hemann, M.T.; Hernando-Monge, E.; Mu, D.; Goodson, S.; Powers, S.; Cordon-Cardo, C.; Lowe, S.W.; Hannon, G.J.; et al. A microrna polycistron as a potential human oncogene. Nature 2005, 435, 828–833, doi:10.1038/nature03552.
- Ota, A.; Tagawa, H.; Karnan, S.; Tsuzuki, S.; Karpas, A.; Kira, S.; Yoshida, Y.; Seto, M. Identification and characterization of a novel gene, c13orf25, as a target for 13q31-q32 amplification in malignant lymphoma. Cancer Res. 2004, 64, 3087–3095, doi:10.1158/0008-5472.CAN-03-3773.
- Mendell, J.T. Miriad roles for the mir-17-92 cluster in development and disease. Cell 2008, 133, 217–222, doi:10.1016/j.cell.2008.04.001.
- Dews, M.; Homayouni, A.; Yu, D.; Murphy, D.; Sevignani, C.; Wentzel, E.; Furth, E.E.; Lee, W.M.; Enders, G.H.; Mendell, J.T.; et al. Augmentation of tumor angiogenesis by a myc-activated microrna cluster. Nat. Genet. 2006, 38, 1060–1065, doi:10.1038/ng1855.
- O'Donnell, K.A.; Wentzel, E.A.; Zeller, K.I.; Dang, C.V.; Mendell, J.T. C-myc-regulated micrornas modulate e2f1 expression. Nature 2005, 435, 839–843, doi:10.1038/nature03677.
- Ivanovska, I.; Ball, A.S.; Diaz, R.L.; Magnus, J.F.; Kibukawa, M.; Schelter, J.M.; Kobayashi, S.V.; Lim, L.; Burchard, J.; Jackson, A.L.; et al. Micrornas in the mir-106b family regulate p21/cdkn1a and promote cell cycle progression. Mol. Cell. Biol. 2008, 28, 2167–2174, doi:10.1128/MCB.01977-07.
- Bloomston, M.; Frankel, W.L.; Petrocca, F.; Volinia, S.; Alder, H.; Hagan, J.P.; Liu, C.G.; Bhatt, D.; Taccioli, C.; Croce, C.M. Microrna expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA 2007, 297, 1901–1908, doi:10.1001/jama.297.17.1901.
- Meng, F.; Henson, R.; Wehbe-Janek, H.; Ghoshal, K.; Jacob, S.T.; Patel, T. Microrna-21 regulates expression of the pten tumor suppressor gene in human hepatocellular cancer. Gastroenterology 2007, 133, 647–658, doi:10.1053/j.gastro.2007.05.022.
- Selcuklu, S.D.; Donoghue, M.T.; Spillane, C. Mir-21 as a key regulator of oncogenic processes. Biochem. Soc. Trans. 2009, 37, 918–925, doi:10.1042/BST0370918.
- Novakova, J.; Slaby, O.; Vyzula, R.; Michalek, J. Microrna involvement in glioblastoma pathogenesis. Biochem. Biophys. Res. Commun. 2009, 386, 1–5, doi:10.1016/j.bbrc.2009.06.034.
- Sana, J.; Hajduch, M.; Michalek, J.; Vyzula, R.; Slaby, O. Micrornas and glioblastoma: Roles in core signalling pathways and potential clinical implications. J. Cell. Mol. Med. 2011, 15, 1636–1644, doi:10.1111/j.1582-4934.2011.01317.x.
- Zhang, Y.; Dutta, A.; Abounader, R. The role of micrornas in glioma initiation and progression. Front. Biosci. 2012, 17, 700–712, doi:10.2741/3952.
- Goke, R.; Barth, P.; Schmidt, A.; Samans, B.; Lankat-Buttgereit, B. Programmed cell death protein 4 suppresses cdk1/cdc2 via induction of p21(waf1/cip1). Am. J. Physiol. Cell. Physiol. 2004, 287, C1541–C1546, doi:10.1152/ajpcell.00025.2004.
- Papagiannakopoulos, T.; Shapiro, A.; Kosik, K.S. Microrna-21 targets a network of key tumor-suppressive pathways in glioblastoma cells. Cancer Res. 2008, 68, 8164–8172, doi:10.1158/0008-5472.CAN-08-1305.
- Gabriely, G.; Wurdinger, T.; Kesari, S.; Esau, C.C.; Burchard, J.; Linsley, P.S.; Krichevsky, A.M. Microrna 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol. Cell. Biol. 2008, 28, 5369–5380, doi:10.1128/MCB.00479-08.
- Gabriely, G.; Yi, M.; Narayan, R.S.; Niers, J.M.; Wurdinger, T.; Imitola, J.; Ligon, K.L.; Kesari, S.; Esau, C.; Stephens, R.M.; et al. Human glioma growth is controlled by microrna-10b. Cancer Res. 2011, 71, 3563–3572, doi:10.1158/0008-5472.CAN-10-3568.
- Ma, L.; Teruya-Feldstein, J.; Weinberg, R.A. Tumour invasion and metastasis initiated by microrna-10b in breast cancer. Nature 2007, 449, 682–688, doi:10.1038/nature06174.
- Baffa, R.; Fassan, M.; Volinia, S.; O'Hara, B.; Liu, C.G.; Palazzo, J.P.; Gardiman, M.; Rugge, M.; Gomella, L.G.; Croce, C.M.; et al. Microrna expression profiling of human metastatic cancers identifies cancer gene targets. J. Pathol. 2009, 219, 214–221, doi:10.1002/path.2586.
- Tan, H.X.; Wang, Q.; Chen, L.Z.; Huang, X.H.; Chen, J.S.; Fu, X.H.; Cao, L.Q.; Chen, X.L.; Li, W.; Zhang, L.J. Microrna-9 reduces cell invasion and e-cadherin secretion in sk-hep-1 cell. Med. Oncol. 2010, 27, 654–660, doi:10.1007/s12032-009-9264-2.
- Ciafre, S.A.; Galardi, S.; Mangiola, A.; Ferracin, M.; Liu, C.G.; Sabatino, G.; Negrini, M.; Maira, G.; Croce, C.M.; Farace, M.G. Extensive modulation of a set of micrornas in primary glioblastoma. Biochem. Biophys. Res. Commun. 2005, 334, 1351–1358, doi:10.1016/j.bbrc.2005.07.030.
- Liu, Y.; Zhao, J.; Zhang, P.Y.; Zhang, Y.; Sun, S.Y.; Yu, S.Y.; Xi, Q.S. Microrna-10b targets e-cadherin and modulates breast cancer metastasis. Med. Sci. Monit. 2012, 18, BR299–BR308, doi:10.12659/MSM.883262.
- Hossain, A.; Kuo, M.T.; Saunders, G.F. Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of aib1 mrna. Mol. Cell. Biol. 2006, 26, 8191–8201, doi:10.1128/MCB.00242-06.
- Cheng, A.M.; Byrom, M.W.; Shelton, J.; Ford, L.P. Antisense inhibition of human mirnas and indications for an involvement of mirna in cell growth and apoptosis. Nucleic Acids Res. 2005, 33, 1290–1297, doi:10.1093/nar/gki200.
- Esau, C.C. Inhibition of microrna with antisense oligonucleotides. Methods 2008, 44, 55–60, doi:10.1016/j.ymeth.2007.11.001.
- Lee, Y.S.; Kim, H.K.; Chung, S.; Kim, K.S.; Dutta, A. Depletion of human micro-rna mir-125b reveals that it is critical for the proliferation of differentiated cells but not for the down-regulation of putative targets during differentiation. J. Biol. Chem. 2005, 280, 16635–16641, doi:10.1074/jbc.M412247200.
- Kloosterman, W.P.; Lagendijk, A.K.; Ketting, R.F.; Moulton, J.D.; Plasterk, R.H. Targeted inhibition of mirna maturation with morpholinos reveals a role for mir-375 in pancreatic islet development. PLoS Biol. 2007, 5, e203, doi:10.1371/journal.pbio.0050203.
- Gantier, M.P.; McCoy, C.E.; Rusinova, I.; Saulep, D.; Wang, D.; Xu, D.; Irving, A.T.; Behlke, M.A.; Hertzog, P.J.; Mackay, F.; et al. Analysis of microrna turnover in mammalian cells following dicer1 ablation. Nucleic Acids Res. 2011, 39, 5692–5703, doi:10.1093/nar/gkr148.
- Lee, Y.; Hur, I.; Park, S.Y.; Kim, Y.K.; Suh, M.R.; Kim, V.N. The role of pact in the rna silencing pathway. EMBO J. 2006, 25, 522–532, doi:10.1038/sj.emboj.7600942.
- Zhang, B.; Farwell, M.A. Micrornas: A new emerging class of players for disease diagnostics and gene therapy. J. Cell. Mol. Med. 2008, 12, 3–21, doi:10.1111/j.1582-4934.2007.00196.x.
- Hutvagner, G.; Simard, M.J.; Mello, C.C.; Zamore, P.D. Sequence-specific inhibition of small rna function. PLoS Biol. 2004, 2, E98, doi:10.1371/journal.pbio.0020098.
- Krutzfeldt, J.; Rajewsky, N.; Braich, R.; Rajeev, K.G.; Tuschl, T.; Manoharan, M.; Stoffel, M. Silencing of micrornas in vivo with “antagomirs”. Nature 2005, 438, 685–689, doi:10.1038/nature04303.
- Esau, C.; Davis, S.; Murray, S.F.; Yu, X.X.; Pandey, S.K.; Pear, M.; Watts, L.; Booten, S.L.; Graham, M.; McKay, R.; et al. Mir-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab. 2006, 3, 87–98, doi:10.1016/j.cmet.2006.01.005.
- Davis, S.; Lollo, B.; Freier, S.; Esau, C. Improved targeting of mirna with antisense oligonucleotides. Nucleic Acids Res. 2006, 34, 2294–2304, doi:10.1093/nar/gkl183.
- Orom, U.A.; Kauppinen, S.; Lund, A.H. Lna-modified oligonucleotides mediate specific inhibition of microrna function. Gene 2006, 372, 137–141, doi:10.1016/j.gene.2005.12.031.
- Nielsen, P.E.; Egholm, M.; Berg, R.H.; Buchardt, O. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 1991, 254, 1497–1500.
- Fabbri, E.; Brognara, E.; Borgatti, M.; Lampronti, I.; Finotti, A.; Bianchi, N.; Sforza, S.; Tedeschi, T.; Manicardi, A.; Marchelli, R.; et al. Mirna therapeutics: Delivery and biological activity of peptide nucleic acids targeting mirnas. Epigenomics 2011, 3, 733–745, doi:10.2217/epi.11.90.
- Torres, A.G.; Fabani, M.M.; Vigorito, E.; Williams, D.; Al-Obaidi, N.; Wojciechowski, F.; Hudson, R.H.; Seitz, O.; Gait, M.J. Chemical structure requirements and cellular targeting of microrna-122 by peptide nucleic acids anti-mirs. Nucleic Acids Res. 2011, 40, 2152–2167.
- Shiraishi, T.; Nielsen, P.E. Cellular Bioavailability of Peptide Nucleic Acids (Pnas) Conjugated to Cell Penetrating Peptides. In Delivery Technologies for Biopharmaceuticals: Peptides, Proteins, Nucleic Acids and Vaccines; Jorgensen, L., Nielsen, H.M., Eds.; John Wiley and Sons Ltd: Hoboken, NJ, USA, 2009.
- Babar, I.A.; Cheng, C.J.; Booth, C.J.; Liang, X.; Weidhaas, J.B.; Saltzman, W.M.; Slack, F.J. Nanoparticle-based therapy in an in vivo microrna-155 (mir-155)-dependent mouse model of lymphoma. Proc. Natl. Acad. Sci. USA 2012, 109, E1695–E1704.
- Ebert, M.S.; Neilson, J.R.; Sharp, P.A. Microrna sponges: Competitive inhibitors of small rnas in mammalian cells. Nat. Methods 2007, 4, 721–726, doi:10.1038/nmeth1079.
- Kluiver, J.; Gibcus, J.H.; Hettinga, C.; Adema, A.; Richter, M.K.; Halsema, N.; Slezak-Prochazka, I.; Ding, Y.; Kroesen, B.J.; van den Berg, A. Rapid generation of microrna sponges for microrna inhibition. PLoS One 2012, 7, e29275, doi:10.1371/journal.pone.0029275.
- Li, T.; Li, D.; Sha, J.; Sun, P.; Huang, Y. Microrna-21 directly targets marcks and promotes apoptosis resistance and invasion in prostate cancer cells. Biochem. Biophys. Res. Commun. 2009, 383, 280–285, doi:10.1016/j.bbrc.2009.03.077.
- Si, M.L.; Zhu, S.; Wu, H.; Lu, Z.; Wu, F.; Mo, Y.Y. Mir-21-mediated tumor growth. Oncogene 2007, 26, 2799–2803, doi:10.1038/sj.onc.1210083.
- Seike, M.; Goto, A.; Okano, T.; Bowman, E.D.; Schetter, A.J.; Horikawa, I.; Mathe, E.A.; Jen, J.; Yang, P.; Sugimura, H.; et al. Mir-21 is an egfr-regulated anti-apoptotic factor in lung cancer in never-smokers. Proc. Natl. Acad. Sci. USA 2009, 106, 12085–12090, doi:10.1073/pnas.0905234106.
- Hatley, M.E.; Patrick, D.M.; Garcia, M.R.; Richardson, J.A.; Bassel-Duby, R.; van Rooij, E.; Olson, E.N. Modulation of k-ras-dependent lung tumorigenesis by microrna-21. Cancer Cell 2010, 18, 282–293, doi:10.1016/j.ccr.2010.08.013.
- Park, J.K.; Lee, E.J.; Esau, C.; Schmittgen, T.D. Antisense inhibition of microrna-21 or -221 arrests cell cycle, induces apoptosis, and sensitizes the effects of gemcitabine in pancreatic adenocarcinoma. Pancreas 2009, 38, e190–e199, doi:10.1097/MPA.0b013e3181ba82e1.
- Basu, A.; Alder, H.; Khiyami, A.; Leahy, P.; Croce, C.M.; Haldar, S. Microrna-375 and microrna-221: Potential noncoding rnas associated with antiproliferative activity of benzyl isothiocyanate in pancreatic cancer. Genes Cancer 2011, 2, 108–119, doi:10.1177/1947601911409212.
- Park, J.K.; Kogure, T.; Nuovo, G.J.; Jiang, J.; He, L.; Kim, J.H.; Phelps, M.A.; Papenfuss, T.L.; Croce, C.M.; Patel, T.; et al. Mir-221 silencing blocks hepatocellular carcinoma and promotes survival. Cancer Res. 2011, 71, 7608–7616, doi:10.1158/0008-5472.CAN-11-1144.
- Ma, L.; Reinhardt, F.; Pan, E.; Soutschek, J.; Bhat, B.; Marcusson, E.G.; Teruya-Feldstein, J.; Bell, G.W.; Weinberg, R.A. Therapeutic silencing of mir-10b inhibits metastasis in a mouse mammary tumor model. Nat. Biotechnol. 2010, 28, 341–347.
- Henry, J.C.; Azevedo-Pouly, A.C.; Schmittgen, T.D. Microrna replacement therapy for cancer. Pharm. Res. 2011, 28, 3030–3042, doi:10.1007/s11095-011-0548-9.
- Behlke, M.A. Chemical modification of sirnas for in vivo use. Oligonucleotides 2008, 18, 305–319, doi:10.1089/oli.2008.0164.
- Choung, S.; Kim, Y.J.; Kim, S.; Park, H.O.; Choi, Y.C. Chemical modification of sirnas to improve serum stability without loss of efficacy. Biochem. Biophys. Res. Commun. 2006, 342, 919–927, doi:10.1016/j.bbrc.2006.02.049.
- Prakash, T.P.; Allerson, C.R.; Dande, P.; Vickers, T.A.; Sioufi, N.; Jarres, R.; Baker, B.F.; Swayze, E.E.; Griffey, R.H.; Bhat, B. Positional effect of chemical modifications on short interference rna activity in mammalian cells. J. Med. Chem. 2005, 48, 4247–4253, doi:10.1021/jm050044o.
- Kitade, Y.; Akao, Y. Micrornas and their therapeutic potential for human diseases: Micrornas, mir-143 and -145, function as anti-oncomirs and the application of chemically modified mir-143 as an anti-cancer drug. J. Pharmacol. Sci. 2010, 114, 276–280, doi:10.1254/jphs.10R12FM.
- Hamm, S.; Latz, E.; Hangel, D.; Muller, T.; Yu, P.; Golenbock, D.; Sparwasser, T.; Wagner, H.; Bauer, S. Alternating 2'-o-ribose methylation is a universal approach for generating non-stimulatory sirna by acting as tlr7 antagonist. Immunobiology 2010, 215, 559–569, doi:10.1016/j.imbio.2009.09.003.
- Chorn, G.; Klein-McDowell, M.; Zhao, L.; Saunders, M.A.; Flanagan, W.M.; Willingham, A.T.; Lim, L.P. Single-stranded microrna mimics. RNA 2012, 18, 1796–1804, doi:10.1261/rna.031278.111.
- Esau, C.C.; Monia, B.P. Therapeutic potential for micrornas. Adv. Drug Deliv. Rev. 2007, 59, 101–114, doi:10.1016/j.addr.2007.03.007.
- Liu, Y.P.; Berkhout, B. Mirna cassettes in viral vectors: Problems and solutions. Biochim. Biophys. Acta 2011, 1809, 732–745.
- McManus, M.T.; Petersen, C.P.; Haines, B.B.; Chen, J.; Sharp, P.A. Gene silencing using micro-rna designed hairpins. RNA 2002, 8, 842–850, doi:10.1017/S1355838202024032.
- Sibley, C.R.; Seow, Y.; Wood, M.J. Novel rna-based strategies for therapeutic gene silencing. Mol. Ther. 2010, 18, 466–476, doi:10.1038/mt.2009.306.
- Grimm, D.; Kay, M.A. Rnai and gene therapy: A mutual attraction. Hematology Am. Soc. Hematol. Educ. Program. 2007, 473–481, doi:10.1182/asheducation-2007.1.473.
- Boudreau, R.L.; Martins, I.; Davidson, B.L. Artificial micrornas as sirna shuttles: Improved safety as compared to shrnas in vitro and in vivo. Mol. Ther. 2009, 17, 169–175, doi:10.1038/mt.2008.231.
- McBride, J.L.; Boudreau, R.L.; Harper, S.Q.; Staber, P.D.; Monteys, A.M.; Martins, I.; Gilmore, B.L.; Burstein, H.; Peluso, R.W.; Polisky, B.; et al. Artificial mirnas mitigate shrna-mediated toxicity in the brain: Implications for the therapeutic development of rnai. Proc. Natl. Acad. Sci. USA 2008, 105, 5868–5873, doi:10.1073/pnas.0801775105.
- Jackson, A.L.; Burchard, J.; Schelter, J.; Chau, B.N.; Cleary, M.; Lim, L.; Linsley, P.S. Widespread sirna “Off-target” Transcript silencing mediated by seed region sequence complementarity. RNA 2006, 12, 1179–1187, doi:10.1261/rna.25706.
- Liu, C.; Kelnar, K.; Liu, B.; Chen, X.; Calhoun-Davis, T.; Li, H.; Patrawala, L.; Yan, H.; Jeter, C.; Honorio, S.; et al. The microrna mir-34a inhibits prostate cancer stem cells and metastasis by directly repressing cd44. Nat. Med. 2011, 17, 211–215, doi:10.1038/nm.2284.
- Tazawa, H.; Tsuchiya, N.; Izumiya, M.; Nakagama, H. Tumor-suppressive mir-34a induces senescence-like growth arrest through modulation of the e2f pathway in human colon cancer cells. Proc. Natl. Acad. Sci. USA 2007, 104, 15472–15477, doi:10.1073/pnas.0707351104.
- Li, L.; Xie, X.; Luo, J.; Liu, M.; Xi, S.; Guo, J.; Kong, Y.; Wu, M.; Gao, J.; Xie, Z.; et al. Targeted expression of mir-34a using the t-visa system suppresses breast cancer cell growth and invasion. Mol. Ther. 2012, 20, 2326–2334, doi:10.1038/mt.2012.201.
- Takamizawa, J.; Konishi, H.; Yanagisawa, K.; Tomida, S.; Osada, H.; Endoh, H.; Harano, T.; Yatabe, Y.; Nagino, M.; Nimura, Y.; et al. Reduced expression of the let-7 micrornas in human lung cancers in association with shortened postoperative survival. Cancer Res. 2004, 64, 3753–3756, doi:10.1158/0008-5472.CAN-04-0637.
- Johnson, C.D.; Esquela-Kerscher, A.; Stefani, G.; Byrom, M.; Kelnar, K.; Ovcharenko, D.; Wilson, M.; Wang, X.; Shelton, J.; Shingara, J.; et al. The let-7 microrna represses cell proliferation pathways in human cells. Cancer Res. 2007, 67, 7713–7722, doi:10.1158/0008-5472.CAN-07-1083.
- Marquez, R.T.; McCaffrey, A.P. Advances in micrornas: Implications for gene therapists. Hum. Gene Ther. 2008, 19, 27–38, doi:10.1089/hum.2007.147.
- Kefas, B.; Godlewski, J.; Comeau, L.; Li, Y.; Abounader, R.; Hawkinson, M.; Lee, J.; Fine, H.; Chiocca, E.A.; Lawler, S.; et al. Microrna-7 inhibits the epidermal growth factor receptor and the akt pathway and is down-regulated in glioblastoma. Cancer Res. 2008, 68, 3566–3572, doi:10.1158/0008-5472.CAN-07-6639.
- Zhang, Y.; Chao, T.; Li, R.; Liu, W.; Chen, Y.; Yan, X.; Gong, Y.; Yin, B.; Qiang, B.; Zhao, J.; et al. Microrna-128 inhibits glioma cells proliferation by targeting transcription factor e2f3a. J. Mol. Med. 2009, 87, 43–51, doi:10.1007/s00109-008-0403-6.
- Jopling, C.L.; Yi, M.; Lancaster, A.M.; Lemon, S.M.; Sarnow, P. Modulation of hepatitis c virus rna abundance by a liver-specific microrna. Science 2005, 309, 1577–1581, doi:10.1126/science.1113329.
- Lanford, R.E.; Hildebrandt-Eriksen, E.S.; Petri, A.; Persson, R.; Lindow, M.; Munk, M.E.; Kauppinen, S.; Orum, H. Therapeutic silencing of microrna-122 in primates with chronic hepatitis c virus infection. Science 2010, 327, 198–201, doi:10.1126/science.1178178.
- Lindow, M.; Kauppinen, S. Discovering the first microrna-targeted drug. J. Cell Biol. 2012, 199, 407–412, doi:10.1083/jcb.201208082.
- Bader, A.G. Mir-34—A microrna replacement therapy is headed to the clinic. Front. Genet. 2012, 3, 120, doi:10.3389/fgene.2012.00120.
- Banks, W.A.; Farr, S.A.; Butt, W.; Kumar, V.B.; Franko, M.W.; Morley, J.E. Delivery across the blood-brain barrier of antisense directed against amyloid beta: Reversal of learning and memory deficits in mice overexpressing amyloid precursor protein. J. Pharmacol. Exp. Ther. 2001, 297, 1113–1121.
- Catuogno, S.; Esposito, C.L.; Quintavalle, C.; Condorelli, G.; de Franciscis, V.; Cerchia, L. Nucleic acids in human glioma treatment: Innovative approaches and recent results. J. Signal. Transduct. 2012, 2012, 735135.
- Du, L.; Kayali, R.; Bertoni, C.; Fike, F.; Hu, H.; Iversen, P.L.; Gatti, R.A. Arginine-rich cell-penetrating peptide dramatically enhances amo-mediated atm aberrant splicing correction and enables delivery to brain and cerebellum. Hum. Mol. Genet. 2011, 20, 3151–3160, doi:10.1093/hmg/ddr217.
- Xia, H.; Mao, Q.; Paulson, H.L.; Davidson, B.L. Sirna-mediated gene silencing in vitro and in vivo. Nat. Biotechnol. 2002, 20, 1006–1010, doi:10.1038/nbt739.
- Lu, P.Y.; Xie, F.; Woodle, M.C. In vivo application of rna interference: From functional genomics to therapeutics. Adv. Genet. 2005, 54, 117–142.
- Tong, A.W. Small rnas and non-small cell lung cancer. Curr. Mol. Med. 2006, 6, 339–349, doi:10.2174/156652406776894554.
- Kota, J.; Chivukula, R.R.; O’Donnell, K.A.; Wentzel, E.A.; Montgomery, C.L.; Hwang, H.W.; Chang, T.C.; Vivekanandan, P.; Torbenson, M.; Clark, K.R.; et al. Therapeutic microrna delivery suppresses tumorigenesis in a murine liver cancer model. Cell 2009, 137, 1005–1017, doi:10.1016/j.cell.2009.04.021.
- Trang, P.; Medina, P.P.; Wiggins, J.F.; Ruffino, L.; Kelnar, K.; Omotola, M.; Homer, R.; Brown, D.; Bader, A.G.; Weidhaas, J.B.; et al. Regression of murine lung tumors by the let-7 microrna. Oncogene 2010, 29, 1580–1587, doi:10.1038/onc.2009.445.
- Bonci, D.; Coppola, V.; Musumeci, M.; Addario, A.; Giuffrida, R.; Memeo, L.; D’Urso, L.; Pagliuca, A.; Biffoni, M.; Labbaye, C.; et al. The mir-15a-mir-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nat. Med. 2008, 14, 1271–1277, doi:10.1038/nm.1880.
- Sicard, F.; Gayral, M.; Lulka, H.; Buscail, L.; Cordelier, P. Targeting mir-21 for the therapy of pancreatic cancer. Mol. Ther. 2013, 21, 986–994, doi:10.1038/mt.2013.35.
- Lee, S.J.; Kim, S.J.; Seo, H.H.; Shin, S.P.; Kim, D.; Park, C.S.; Kim, K.T.; Kim, Y.H.; Jeong, J.S.; Kim, I.H. Over-expression of mir-145 enhances the effectiveness of hsvtk gene therapy for malignant glioma. Cancer Lett. 2012, 320, 72–80, doi:10.1016/j.canlet.2012.01.029.
- Wang, X.; Han, L.; Zhang, A.; Wang, G.; Jia, Z.; Yang, Y.; Yue, X.; Pu, P.; Shen, C.; Kang, C. Adenovirus-mediated shrnas for co-repression of mir-221 and mir-222 expression and function in glioblastoma cells. Oncol. Rep. 2011, 25, 97–105.
- Pramanik, D.; Campbell, N.R.; Karikari, C.; Chivukula, R.; Kent, O.A.; Mendell, J.T.; Maitra, A. Restitution of tumor suppressor micrornas using a systemic nanovector inhibits pancreatic cancer growth in mice. Mol. Cancer Ther. 2011, 10, 1470–1480, doi:10.1158/1535-7163.MCT-11-0152.
- Trang, P.; Wiggins, J.F.; Daige, C.L.; Cho, C.; Omotola, M.; Brown, D.; Weidhaas, J.B.; Bader, A.G.; Slack, F.J. Systemic delivery of tumor suppressor microrna mimics using a neutral lipid emulsion inhibits lung tumors in mice. Mol. Ther. 2011, 19, 1116–1122, doi:10.1038/mt.2011.48.
- Costa, P.M.; Cardoso, A.L.; Mendonca, L.S.; Serani, A.; Custodia, C.; Conceicao, M.; Simoes, S.; Moreira, J.N.; Pereira de Almeida, L.; Pedroso de Lima, M.C. Tumor-targeted chlorotoxin-coupled nanoparticles for nucleic acid delivery to glioblastoma cells: A promising system for glioblastoma treatment. Mol. Ther. Nucleic Acids 2013, 2, e100, doi:10.1038/mtna.2013.30.
- Yang, Y.P.; Chien, Y.; Chiou, G.Y.; Cherng, J.Y.; Wang, M.L.; Lo, W.L.; Chang, Y.L.; Huang, P.I.; Chen, Y.W.; Shih, Y.H.; et al. Inhibition of cancer stem cell-like properties and reduced chemoradioresistance of glioblastoma using microrna145 with cationic polyurethane-short branch pei. Biomaterials 2012, 33, 1462–1476, doi:10.1016/j.biomaterials.2011.10.071.
- Ren, Y.; Kang, C.S.; Yuan, X.B.; Zhou, X.; Xu, P.; Han, L.; Wang, G.X.; Jia, Z.; Zhong, Y.; Yu, S.; et al. Co-delivery of as-mir-21 and 5-fu by poly(amidoamine) dendrimer attenuates human glioma cell growth in vitro. J. Biomater. Sci. Polym. Ed. 2010, 21, 303–314, doi:10.1163/156856209X415828.
- A Multicenter Phase I Study of MRX34, MicroRNA miR-RX34 Liposome Injectable Suspension. Available online: http://clinicaltrials.gov/ct2/show/NCT01829971/ (accessed on 23 September 2013).
- Guan, J.; Fujimoto, K.L.; Sacks, M.S.; Wagner, W.R. Preparation and characterization of highly porous, biodegradable polyurethane scaffolds for soft tissue applications. Biomaterials 2005, 26, 3961–3971, doi:10.1016/j.biomaterials.2004.10.018.
- Liu, X.Y.; Ho, W.Y.; Hung, W.J.; Shau, M.D. The characteristics and transfection efficiency of cationic poly (ester-co-urethane)—Short chain pei conjugates self-assembled with DNA. Biomaterials 2009, 30, 6665–6673, doi:10.1016/j.biomaterials.2009.08.052.
- Bala, I.; Hariharan, S.; Kumar, M.N. Plga nanoparticles in drug delivery: The state of the art. Crit. Rev. Ther. Drug Carrier Syst. 2004, 21, 387–422, doi:10.1615/CritRevTherDrugCarrierSyst.v21.i5.20.
- Pettit, M.W.; Griffiths, P.; Ferruti, P.; Richardson, S.C. Poly(amidoamine) polymers: Soluble linear amphiphilic drug-delivery systems for genes, proteins and oligonucleotides. Ther. Deliv. 2011, 2, 907–917, doi:10.4155/tde.11.55.
- D’Emanuele, A.; Attwood, D. Dendrimer-drug interactions. Adv. Drug Deliv. Rev. 2005, 57, 2147–2162, doi:10.1016/j.addr.2005.09.012.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).