Micro-RNAs (miRNAs) are endogenous, evolutionarily conserved, non-coding, RNAs of about 20-25 nucleotides in length. They control cell fate via cell proliferation, differentiation, apoptosis and stress response. They regulate gene expression by primarily disrupting mRNA translation and stability, or by modulating the transcription of target messenger RNAs. The expression of miRNA has been shown to be deregulated in various human diseases, thus making them novel diagnostic biomarkers and therapeutic targets. It is challenging to target miRNAs with small molecules; different groups have identified small molecule modulators of miRNA by targeting various pathways. In the present review, we discuss about the role of micro-RNAs in cancer, and strategies for modulating their expression, which can be used to achieve therapeutic outcomes.

Pamarthy D, Chowdhury D, Bhadra U, Bhadra MP (2016) Small Molecule Modulators of Micro RNA and Their Role in Cancer Therapy. JSM Biotechnol Bioeng 3(5): 1070.

MiRNAs are short, non-coding RNAs that regulate gene expression at post-transcriptional level [1-3]. They originate via transcription of a miRNA gene by RNA polymerase II. Subsequently they undergo a dual processing step, where an initial nascent transcript folds over itself, forming a secondary hairpin structure, called long primary miRNA (pri-miRNA) [4]. This is then cleaved by the RNase endonuclease III, Drosha, along with a microprocessor part, DGCR8, forming a precursor sequence, called pre-miRNA, of about 70 nucleotides in length [5- 7]. The pre-miRNA is transported to the cytoplasm via Exportin 5 and RanGTP [8,9]. A double stranded RNA-specific cytoplasmic nuclease, called Dicer causes a cleavage to form a 22 nucleotidelong double stranded RNA transcript (dsRNA), following which, the guide strand, involving the RNA-induced silencing complex (RISC) targets the 3′-untranslated region (UTR) of messenger RNAs [10]. The final result is decrease in target protein levels.

Imbalance in the levels of miRNA can destabilize the entire cellular machinery, as a single miRNA can regulate hundreds of mRNA at the same time. Many genes can be regulated simultaneously as their nucleotide pairing with miRNAs is imperfect [11]. In case of human cancer, these genes are responsible for various cancer associated pathways, such as tumor initiation, development, invasion and metastasis. Owing to their aberrant expression, modulation of miRNA levels is of therapeutic potential. miRNAs can be divided into two main groups based on their expression levels: tumor suppressive miRNAs (ts-miRNAs) and oncomiRs. Ts-miRNAs are down regulated and target tumors suppressor genes and oncomiRs have increased expression, targeting oncogenes (Figure 1). In addition to their traditional roles, miRNAs also cause an increase in translation during various cellular processes. For example, human microRNA, miR-369-3 is involved in the association of argonaute (AGO) and fragile X mental retardationrelated protein 1(FXR1), [proteins which are associated with micro-ribonucleoproteins (microRNPs)] with AREs (AU-rich elements) to activate translation. Additionally, microRNAs Let-7 and synthetic microRNA miRcxcr4 induce translation upregulation of target mRNA upon cell cycle arrest; however they repress translation in proliferating cells [12]. miR-15b represses WEE1 (a key mammalian cell cycle regulator) protein during G1 and S phase. Regulatory factors such as CPEB1 (cytoplasmic polyadenylation element-binding protein) also bind to mRNA 3´UTR during post transcriptional repression. Intriguingly, both factors lose their inhibitory activity at G2/M transition, when WEE1 expression is maximal and activate WEE1 translation in a synergistic manner [13]. Another miRNA, miR-276 is involved in the upregulation of transcription coactivator gene (brm), through a process that depends on secondary structure of brm, involving a stem-loop around the binding site of miR-276 [14]. miR-328 upregulates CEBPA (CCAAT-enhancer binding protein alpha) by releasing it from heterogenous ribonucleo protein mediated translation inhibition [15]. miR-373 induces transcription of E-cadherin by targeting promoter sequences [16]. It has also been reported that nuclear entry of miRNAs are necessary for upregulation of genes in some cases [17]. miRNA was also found to function endogenously, activating Cyclin B1 (Ccnb1) expression in mouse cells, thus manipulating in vivo tumor development and growth. Three more miRNAs were found, miR744, miR-1186 and miR-466d-3p that induce Ccnb1 expression in mouse cell lines [18].

MicroRNAs in cancer

Cancer is a complex disease involving changes in gene expression. In recent years there has been tremendous interest in investigating the role of miRNAs in cancer. miRNA expression profiling has been extensively studied and taken into cancer clinics, to be used as a diagnostic and prognostic biomarker, in order to monitor tumor initiation, progression and response to treatment [19]. The roles played by miRNAs have been reported in different types of cancers, such as breast, colon, gastric, lung, prostate and thyroid cancers [20-24]. The role of miRNA in cancer has been extensively studied, as evidenced by peer-reviewed scientific literature covering nearly 23,467 hits on Pubmed, as of November 2016.

miRNAs have either oncogenic or tumor suppressor function and their expression is globally suppressed in tumor cells, compared to normal tissues. miRNA dysregulation in cancer was first reported in CLL (chronic lymphocytic leukemia) in 2002. A cluster of two miRNAs, namely miR-15 and miR-16 were identified at 13q14.3 chromosomal region, which is frequently deleted. This deletion was partly responsible for higher expression of miR-15/16 anti-apoptotic target, B cell lymphoma 2 (BCL2). miRNAs can act as tumor suppressors, such as let-7 family of miRNAs that target critical oncogenes like RAS family members (HRAS, KRAS and NRAS) and MYC. The differential pattern of miRNA expression in cancer allows their use as diagnostic markers, which correlates with disease progression.

Another miRNA, miR-34 consists of a family of three miRs, namely miR-34a, miR-34b, and miR-34c, which have presumably tissue-specific functions, even though they are direct transcriptional targets of the onco-suppressor p53, whose expression is greatly affected by DNA damage and oncogenic stress. Thus miR-34 family contributes to arrest of cell proliferation and induction of apoptosis, by targeting c-MYC, CDK6 and c-MET [25]. Di Martino et al., have reported the molecular effects induced by enforced expression of miR-34a on multiple myeloma (MM) cells, showing time-dependent modulation of several signalling pathways, controlling cell proliferation and apoptosis. This is the first group to have reported the role of miR-34a in the pathogenesis of MM, through its downregulation in a wide series of MM samples [26,27]. The most affected pathway was the Erk/ Akt dependent pathway [27]. The researchers showed that miR34a induces sequential down modulation of both Erk and Akt activity, followed by pro-caspase-6 and -3 cleavage and apoptosis induction in MM cells. Previously, the same scientists have also shown the potential of miR-34a treatment in downregulation of Bcl-2 and NOTCH1, and in induction of apoptosis, both in vitro and in a xenograft mouse model [26]. Di Martino et al., tested the efficiency of stable nucleic acid lipid particles (SNALPs) in delivering miR-34a in vivo [27]. SNALPs have high serum stability and are an attractive option to translate miR based therapies in the clinic. According to these findings, a liposome-carried form of miR-34a is under investigation in Phase I clinical trial in patients affected by several malignancies, including MM (clinicaltrials. gov: A multicenter phase I study of MRX34, miRNA miR-RX34 liposomal injection NCT01829971). Other recent studies explore the blocking of Bcl-2 by vaccinia virus-miR-34a, which increases release of cytochrome C from mitochondria and synergistically amplifies the antitumor effects of Smac-induced cell apoptosis. This is the first study to utilize oncolytic vaccinia virus as vector for miR-34a or Smac expression for treating MM, and lays groundwork for future clinical therapy [28]. Finally, miR-34a has also been associated with regulation of cancer stem cells function in various cancer types, including prostate cancer [29], pancreatic cancer [30], breast cancer [31], and glioblastoma [32].

miRNAs can also function as oncogenes. An oncogenic cluster of miRNAs, the polycistron, miR-17-92, comprising of a cluster of seven miRNAs: miR-17-5p, miR-17-3p, miR18a, miR-19a, miR19b-1, miR-20, and miR-92-1, was found to be overexpressed in many lymphoma samples compared to normal tissues [33]. miR-21 was found to be strongly overexpressed in highly malignant glioblastoma tumor tissues [34]. A knockdown of miR21 in cultured glioblastoma cells activated caspases and led to cell death via an apoptotic pathway. Further, a comprehensive analysis of miR-21 in solid tumors showed overexpression, that is common to six types of cancer that were studied, namely breast, colon, lung, pancreas, prostate, and stomach [35].

Other studies have shown the aberrant expression of miRNAs in brain tumors. The analysis of 245 miRNAs in glioblastoma multiforme showed that miR-221 was highly upregulated. Reports showed that miR-221 and miR-222 are highly expressed in glioblastoma and directly target p27Kip1, a negative regulator of the cell cycle [36]. miRNAs 221/222 are of interest as they are strongly upregulated in a variety of solid and hematologic malignancies. In case of Breast Cancer (BC) miR-221 mainly supports tumor growth and progression. In basal-like BC, miR221/222 is highly expressed and promotes S-phase entry, cell migration and invasion through the inhibition of suppressor of cytokine signaling 1 (SOCS1) and cyclin-dependent kinase inhibitor 1B (CDKN1B) [37]. Upregulation of miR-221/222 is also relevant in triple negative BC, via cell cycle regulation and inhibition of apoptosis [38]. These data suggest that targeting miR-221 may be of value for the therapy of BC. Tanaka R et al. [39], have recently shown that Metformin, an anti-diabetic drug causes G1 phase arrest by downregulating miR-221, followed by rescue of p27 checkpoint and enhancement of TRAIL sensitivity through DR5 upregulation in pancreatic cancer cells. In case of transgenic mice, liver tumors ranging from typical adenomas to HCC (hepatocellular carcinomas) - like lesions, develop with selective upregulation of miR-221. In these tumors, targets of miR-221 such as cell cycle inhibitors, p27, p57 and pro-apoptotic proteins were found to be downregulated. When transgenic mice were intravenously treated with 2´-O-methyl modified oligonucleotides targeting miR-221, tumor growth was inhibited and no systemic toxicity was observed [40]. miR-221/222 also showed tumor suppressor activity in Gastrointestinal Stromal Tumor (GIST) cell lines, where miR-221/222 transfection promoted apoptosis through inhibition of KIT expression and activation of caspase 3 and 7 [41]. In MM cells highly expressing miR-221/222, enforced expression of miR-221/222 inhibitors triggered in vitro anti-proliferative effects, along with upregulation of miR-221/222 targets such as p27Kip1, PUMA, PTEN and p57Kip2. Contrarily, in MM with low basal miR221/222, transfection of miR-221/222 mimics increased S phase and downregulated p27Kip1 protein expression. miR-221/222 inhibitors were also evaluated in MM Xenografts in SCID/NOD mice. Significant anti-tumor activity was observed along with upregulation of established protein targets in tumors retrieved from animals [42].

Modulation of microRNA expression

MicroRNAs regulate the stability and translation of messenger RNA (mRNA), consequently controlling protein synthesis and gene expression. They control these processes at posttranscriptional level by complementary binding to the sequences in the 3’-untranslated region of mRNA, and play a pivotal role in cell fate. Changes in miRNA expression are associated with several human diseases, including cancer, thus making them attractive therapeutic targets. However there are challenges to achieve therapeutic effects, using miRNA. Identification of molecules that regulate loss or gain of function of miRNA and their efficient delivery into the cell are two of the major challenges.

OncomiRs are known to have links to the pathogenesis and aggressiveness of cancer. Large-expression screens comparing tumor versus normal tissues are useful for identifying unique miRNA signatures and can be used as attractive anti-cancer therapeutic targets [43-45]. The approach to miRNA based therapeutics involves inhibition of potent cellular targets. For example, miR-21 targets PTEN and PDCD4 [46-49]. Likewise, miR-155 blocks the translation of CEBPβ, IL17RB, PCCD4, TCF12, ZNF652, which are tumor suppressor genes [35,50]. Additionally, oncogenic miRNA targets have been validated in similar patient populations. The major obstacle in this area is finding an effective delivery mechanism, such as nano-particles, liposomes and peptides, majority of which have proven to be ineffective or toxic [51,52].

miRNA and cancer therapy

For over a decade, miRNAs have been hallmarks of cancer. OncomiRS have been profiled and shown to be causative factors in the activation of oncogenic pathways leading to cancer. Hence it is important to develop strategies to efficiently inhibit their expression. Use of small molecule inhibitors (SMIRs) presents an effective strategy to target specific miRNAs. Other methods of miRNA inhibition are based on antisense oligonucletides (antimiRs), locked nucleic acids (LNA), LNA-antimiR constructs, antagomirs, miRNA sponges, ribozymes/DNAzymes, small interfering RNAs (siRNAs) and short hairpin RNAs (shRNA). The discovery and usage of these strategies for cancer therapy were found to be exciting. However, there are challenges in delivery of these small-molecules, besides the pharmacodynamic and pharmacokinetic properties. Due to challenges in the delivery of these molecules, new approaches to target oncomiRs have to be discovered. The first miRNA molecule to reach clinical trials is Miravirsen, developed by Santaris Pharma A/S, a locked nucleic acid targeting miR-122. It is useful for the treatment of HCV (hepatitis C) infection, which increases the chance of patients developing Hepatocellular carcinoma. Miravirsen completed a phase 2a study wherein a patient reached an undetectable level of HCV-RNA and robust activity was exhibited against viral load [53,54].

Since miRNAs are pivotal in cancer, they have been associated with every hallmark of cancer. Due to the challenges involved in using nucleotide analogs to target miRNAs, the development of small-molecule drugs targeting specific miRNAs was proposed to be a promising approach. The interaction of small molecules with miRNA was termed as “SMIR” by Melo and Calin et al. [55,56], commonly referred as small molecule inhibitors of specific miRNAs. This opened doors for a very specific, targeted cancer therapy. The SMIR-concept is attractive, as it takes shorter time in drug development and also reduces the time for approval and production, thus reducing the overall cost of drug discovery. However this approach is also challenging and risky. RNA transcripts, in the past, were not explored fully as drug targets, due to their electronegative charge and flexible structure. Additionally, X-Ray crystallography as well as Nuclear Magnetic Resonance structures for miRNAs were not well developed. The lower availability of miRNA-Dicer or RISC complex structures made drug discovery difficult. If these barriers are overcome, the SMIR approach will result in having effective drug delivery to patients.

More recently, there is advancement in the development of efficient LNAs, targeting various cancers. miR-221/222 antisense LNA oligonucleotides reduce tumor growth by increasing intratumor p27Kip1 protein expression in prostate carcinoma [57]. Other researchers showed the possibility to extend the results to human studies, prompting investigators to develop a novel 13-mer locked nucleic acid (LNA)-chemically modified miR-221 inhibitor, a fully phosphorothioate (PS)-modified backbone, the LNAi-miR-221 [58]. LNA modifications conferred prolonged effects and higher stability. This novel approach was also prompted by recent successful treatment of chronic hepatitis C patients with miR-122 LNA inhibitor [59]. Maria Eugenia Gallo Cantafio et al., demonstrated short half-life, optimal tissue biovailability and minimal urine excretion of LNA-i-miR-221 in mice and monkeys. LNA-i-miR-221 was still detectable in mice vital organs and in xenografted tumors, upto 3 weeks, along with p27 target upregulation. No toxicity in the pilot study with monkey model was observed [60]. In summary, these data suggest a prominent role of miR-221/222 in the development and progression of solid and hematological tumors. The recent findings on the use of selective inhibitors, as LNA-i-miR-221, both in vitro and in vivo are opening a new avenue for the design of miRNA-based therapies to be tested in clinical trials.

RNA as a target

RNA is an attractive target for drug development as it folds into three dimensional structures and is involved in various cellular processes [61,62]. Two main targeting strategies are (1) Oligonucleotides and (2) Small molecules. Due to ease of base-pairing, as per Watson-Crick rules, RNAs can be targeted by oligonucleotides. They are however high molecular weight compounds that cause difficulties in delivery into the cells. Modifications of oligonucleotides have helped traverse the cell membrane and reach the diseased tissues. Oligonucleotides can be synthesized to block translation, inhibit toxic RNA-protein interactions and avoid cryptic splice sites associated with disease [63]. Studies have shown that RNA can be used as a therapeutic target via small molecule interactions [64-66]. Small molecules have been designed, to target RNA repeats in genetic diseases [67-73] and miRNA precursors involved in cancer and other diseases [74-76].

Development of Oligonucleotides

Antisense oligonucleotides (ASOs) were first developed by Zamecnik et al., to target Rous sarcoma virus [77,78]. Complementary DNAs were used to bind to the virus and reduce the production of its RNA. Studies showed that DNA oligonucleotides form DNA-RNA hybrids in cells, which then recruit RNase H and result in cleavage of the RNA strand [79]. Modified oligonucleotides were made to improve stability in the presence of endonucleases, while retaining the RNase-dependent activity [80]. Site-specific modifications in the backbone and sugar moiety were created to increase the potential of these molecules to treat human diseases [81-83].

The most common accepted mechanism of action of ASOs, involves formation of mRNA-ASO duplex, through Watson-Crick pairing, leading to RNase-dependent cleavage of target mRNA [84-86]. Other mechanisms involve blockage of mRNA transport, modulation of splicing, translational arrest and formation of a triple helical structure through ASO binding to double stranded DNA resulting in inhibition of transcription. Chemical modifications of ASO drugs will increase in vivo half life, improve distribution to diseased tissue, increase potency and reduce toxic effects. Better understanding of importance of therapeutic target, dose optimization and scheduling, increasing trials with tumors that are sensitive to inhibition of relevant target and their use as combination therapy will increase the likelihood of success.

Small molecules to target RNA

Targeting RNA with small molecules is a challenging task, due to binding problems, non-specific interactions, and structural redundancy. A major advantage is that RNA folds into different structures and is composed of base pairs, and non-canonically paired regions, such as hairpins, loops and bulges. Base-paired regions are common to all RNAs, whereas the latter are unique to a single RNA or a smaller subset. A lead identification strategy, named Inforna [74,75] was proposed, that compares secondary structural elements in a target RNA to known RNA motif-small molecular interactions that are highly selective. Inforna has reported the design of small molecule leads that target RNAs involved in microsatellite disorders and miRNAs linked to cancer. Other methods in the design of small molecules to target RNA are structure-dependent. This has proven useful to generate molecules mimicking the binding of proteins to RNA. Another method is docking of small molecules into RNA, using NMR spectroscopy and molecular dynamics simulations [87,88].

There is an increased interest in RNA-drug discovery, owing to the roles played by RNA in healthy and diseased tissues. Precise medicines can be developed, that target RNA and affect cellular function. Oligonucleotides as well as small molecules are of therapeutic potential. There is need, not only for making effective RNA inhibiting molecules, but to validate them. Rational computational methods have been validated, that enable functional studies on lead compounds, by predicting 3D structures of miRNAs. Undoubtedly miRNAs are being considered as potential molecules for targeting in cancer.

A more precise way to silence specific miRNAs, would be to find a compound that binds to the primary, precursor or mature sequence of a miRNA in a specific manner. For instance, miR-21 is overexpressed in breast, ovary, cervix, colon, lung, liver, brain, esophagus, prostate, pancreas and thyroid cancers. A SMIR that is specific to miR-21 is most likely to treat patients with these cancers via expression of OncomiR-21, as it negatively the tumor suppressive targets PTEN, PDCD4 and RECK. However we should also expect off-target effects, in both tissue of interest and throughout the body. Therefore newly discovered SMIRs have to undergo thorough in vitro and in vivo testing, before being released for patient use.

1. Ambros V. MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell. 2003; 113: 673-676.

2. Lai EC. microRNAs: runts of the genome assert themselves. Curr Biol. 2003; 13: 925-936.

3. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004; 116: 281-297.

4. Zeng Y, Cullen BR. Sequence requirements for micro RNA processing and function in human cells. RNA. 2003; 9: 112-123.

5. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003; 425: 415-419.

6. Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, et al. The Microprocessor complex mediates the genesis of microRNAs. Nature. 2004; 432: 235-240.

7. Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 2004; 18: 3016- 3027.

8. Yi R, Qin Y, Macara IG, Cullen BR. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 2003; 17: 3011-3016.

9. Lund E, Güttinger S, Calado A, Dahlberg JE, Kutay U. Nuclear export of microRNA precursors. Science. 2004; 303: 95-98.

10. Hutvágner G, McLachlan J, Pasquinelli AE, Bálint E, Tuschl T, Zamore PD. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science. 2001; 293: 834- 838.

11. He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004; 5: 522-31.

12. Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: microRNAs can up-regulate translation. Science. 2007; 318: 1931-1934.

13. Kratassiouk G, Pritchard LL, Cuvellier S, Vislovukh A, Meng Q, Groisman R, et al. The WEE1 regulators CPEB1 and miR-15b switch from inhibitor to activators at G2/M. Cell Cycle. 2016; 15: 667-677.

14. He J, Chen Q, Wei Y, Jiang F, Yang M, Hao S, et al. MicroRNA-276 promotes egg-hatching synchrony by up-regulating brm in locusts. Proc Natl Acad Sci U S A. 2016; 113: 584-589.

15. Eiring AM, Harb JG, Neviani P, Garton C, Oaks JJ, Spizzo R, et al. miR328 functions as an RNA decoy to modulate hnRNP E2 regulation of mRNA translation in leukemic blasts. Cell. 2010; 140: 652-665.

16. Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R. MicroRNA-373 induces expression of genes with complementary promoter sequences. Proc Natl Acad Sci U S A. 2008; 105: 1608-1613.

17. Mortensena RD, Serraa M, Steitzb JA, Vasudevan S. Posttranscriptional activation of gene expression in Xenopus laevis oocytes by microRNAprotein complexes (microRNPs). Proc Natl Acad Sci U S A. 2011; 108: 8281-8286.

18. Huang V, Place RF, Portnoy V, Wang J, Qi Z, Jia Z, et al. Upregulation of Cyclin B1 by miRNA and its implications in cancer. Nucleic Acids Res. 2012; 40: 1695-1707.

19. Reddy KB. MicroRNA (miRNA) in cancer. Cancer Cell Int. 2015; 15: 38.

20. He H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S, et al. The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci U S A. 2005; 102: 19075-19080.

21. Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 2005; 65: 7065-7070.

22. Michael MZ, O’Connor SM, van Holst Pellekaan NG, Young GP, James RJ. Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol Cancer Res. 2003; 1: 882-891.

23. Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H, 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.

24. Di Leva G, Croce CM. The Role of microRNAs in the Tumorigenesis of Ovarian Cancer. Front Oncol. 2013; 3: 153.

25. Hermeking H. The miR-34 family in cancer and apoptosis. Cell Death Differ. 2010; 17: 193-199.

26. Di Martino MT, Leone E, Amodio N, Foresta U, Lionetti M, Pitari MR, et al. Synthetic miR-34a mimics as a novel therapeutic agent for multiple myeloma: in vitro and in vivo evidence. Clin Cancer Res. 2012; 18: 6260-6270.

27. Di Martino MT, Campani V, Misso G, Gallo Cantafio ME, Gullà A, Foresta U, et al. In vivo activity of miR-34a mimics delivered by stable nucleic acid lipid particles (SNALPs) against multiple myeloma. PLoS One. 2014; 9: 90005.

28. Lei W, Wang S, Yang C, Huang X, Chen Z, He W, et al. Combined expression of miR-34a and Smac mediated by oncolytic vaccinia virus synergistically promote anti-tumor effects in Multiple Myeloma. Sci Rep. 2016; 6: 32174.

29. Liu C, Kelnar K, Liu B, Chen X, Calhoun-Davis T, Li H, et al. The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44. Nat Med. 2011; 17: 211-215.

30. Nalls D, Tang SN, Rodova M, Srivastava RK, Shankar S. Targeting epigenetic regulation of miR-34a for treatment of pancreatic cancer by inhibition of pancreatic cancer stem cells. PLoS One. 2011; 6: 24099.

31. Stankevicins L, Almeida da Silva AP, Ventura Dos Passos F, Dos Santos Ferreira E, Menks Ribeiro MC, G David M, et al. MiR-34a is up-regulated in response to low dose, low energy X-ray induced DNA damage in breast cells. Radiat Oncol. 2013; 8: 231.

32. Guessous F, Zhang Y, Kofman A, Catania A, Li Y, Schiff D, et al. microRNA-34a is tumor suppressive in brain tumors and glioma stem cells. Cell Cycle. 2010; 9: 1031-1036.

33. He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S, et al. A microRNA polycistron as a potential human oncogene. Nature. 2005; 435: 828-833.

34. Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 2005; 65: 6029-6033.

35. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006; 103: 2257-2261.

36. Gillies JK, Lorimer IA. Regulation of p27Kip1 by miRNA 221/222 in glioblastoma. Cell Cycle. 2007; 6: 2005-2009.

37. Li Y, Liang C, Ma H, Zhao Q, Lu Y, Xiang Z, et al. miR-221/222 promotes S-phase entry and cellular migration in control of basal-like breast cancer. Molecules. 2014; 19: 7122-7137.

38. Nassirpour R, Mehta PP, Baxi SM, Yin MJ. miR-221 promotes tumorigenesis in human triple negative breast cancer cells. PLoS One. 2013; 8: 62170.

39. Tanaka R, Tomosugi M, Horinaka M, Sowa Y, Sakai T. Metformin Causes G1-Phase Arrest via Down-Regulation of MiR-221 and Enhances TRAIL Sensitivity through DR5 Up-Regulation in Pancreatic Cancer Cells. PloS One. 2015; 10: 0125779.

40. Callegari E, Elamin BK, Giannone F, Milazzo M, Altavilla G, Fornari F, et al. Liver tumorigenicity promoted by microRNA-221 in a mouse transgenic model. Hepatology. 2012; 56: 1025-1033.

41. Ihle MA, Trautmann M, Kuenstlinger H, Huss S, Heydt C, Fassunke J, et al. miRNA-221 and miRNA-222 induce apoptosis via the KIT/AKT signalling pathway in gastrointestinal stromal tumours. Mol Oncol. 2015; 9: 1421-1433.

42. Di Martino MT, Gullà A, Cantafio ME, Lionetti M, Leone E, Amodio N, et al. In vitro and in vivo anti-tumor activity of miR-221/222 inhibitors in multiple myeloma. Oncotarget. 2013; 4: 242-255.

43. Ferdin J, Kunej T, Calin GA. Non-coding RNAs: identification of cancerassociated microRNAs by gene profiling. Technol Cancer Res Treat. 2010; 9: 123-138.

44. Munker R, Calin GA. MicroRNA profiling in cancer. Clin Sci (Lond). 2011; 121: 141-158.

45. Di Leva G, Croce CM. The Role of microRNAs in the Tumorigenesis of Ovarian Cancer. Front Oncol. 2013; 3: 153.

46. Lou Y, Yang X, Wang F, Cui Z, Huang Y. MicroRNA-21 promotes the cell proliferation, invasion and migration abilities in ovarian epithelial carcinomas through inhibiting the expression of PTEN protein. Int J Mol Med. 2010; 26: 819-827.

47. Yan LX, Huang XF, Shao Q, Huang MY, Deng L, Wu QL, et al. MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA. 2008; 14: 2348-2360.

48. Gumireddy K, Young DD, Xiong X, Hogenesch JB, Huang Q, Deiters A. Small-molecule inhibitors of microrna miR-21 function. Angew Chem Int Ed Engl. 2008; 47: 7482-7484.

49. 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.

50. Czyzyk-Krzeska MF, Zhang X. MiR-155 at the heart of oncogenic pathways. Oncogene. 2014; 33: 677-678.

51. Garzon R, Marcucci G, Croce CM. Targeting microRNAs in cancer: rationale, strategies and challenges. Nat Rev Drug Discov. 2010; 9: 775-789.

52. Pecot CV, Calin GA, Coleman RL, Lopez-Berestein G, Sood AK. RNA interference in the clinic: challenges and future directions. Nat Rev Cancer. 2011; 11: 59-67.

53. Lindow M, Kauppinen S. Discovering the first microRNA-targeted drug. J Cell Biol. 2012; 199: 407-412.

54. Janssen HL, Reesink HW, Lawitz EJ, Zeuzem S, Rodriguez-Torres M, Patel K, et al. Treatment of HCV infection by targeting microRNA. N Engl J Med. 2013; 368: 1685-1694.

55. Zhang S, Chen L, Jung EJ, Calin GA. Targeting microRNAs with small molecules: from dream to reality. Clin Pharmacol Ther. 2010; 87: 754- 758.

56. Melo S, Villanueva A, Moutinho C, Davalos V, Spizzo R, Ivan C, et al. Small molecule enoxacin is a cancer-specific growth inhibitor that acts by enhancing TAR RNA-binding protein 2-mediated microRNA processing. Proc Natl Acad Sci U S A. 2011; 108: 4394-4399.

57. Galardi S, Mercatelli N, Giorda E, Massalini S, Frajese GV, Ciafrè SA, et al. miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27Kip1. J Biol Chem. 2007; 282: 23716-23724.

58. Di Martino MT, Gullà A, Gallo Cantafio ME, Altomare E, Amodio N, Leone E, et al. In vitro and in vivo activity of a novel locked nucleic acid (LNA)-inhibitor-miR-221 against multiple myeloma cells. PLoS One. 2014; 9: 89659.

59. Janssen HL, Reesink HW, Lawitz EJ, Zeuzem S, Rodriguez-Torres M, Patel K, et al. Treatment of HCV infection by targeting microRNA. N Engl J Med. 2013; 368: 1685-1694.

60. Gallo Cantafio ME, Nielsen BS, Mignogna C, Arbitrio M, Botta C, Frandsen NM, et al. Pharmacokinetics and Pharmacodynamics of a 13-mer LNA-inhibitor-miR-221 in Mice and Non-human Primates. Mol Ther Nucleic Acids. 2016; 5.

61. Batey RT, Rambo RP, Doudna JA. Tertiary Motifs in RNA Structure and Folding. Angew Chem Int Ed Engl. 1999; 38: 2326-2343.

62. Doudna JA. Structural genomics of RNA. Nat Struct Biol. 2000; 7: 954- 956.

63. Sierakowska H, Agrawal S, Kole R. Antisense oligonucleotides as modulators of pre-mRNA splicing. Methods Mol Biol. 2000; 133: 223- 233.

64. Guan L, Disney MD. Recent advances in developing small molecules targeting RNA. ACS Chem Biol. 2012; 7: 73-86.

65. Gallego J, Varani G. Targeting RNA with small-molecule drugs: therapeutic promise and chemical challenges. Acc Chem Res. 2001; 34: 836-843.

66. Thomas JR, Hergenrother PJ. Targeting RNA with small molecules. Chem Rev. 2008; 108: 1171-1224.

67. Gareiss PC, Sobczak K, McNaughton BR, Palde PB, Thornton CA, Miller BL. Dynamic combinatorial selection of molecules capable of inhibiting the (CUG) repeat RNA-MBNL1 interaction in vitro: discovery of lead compounds targeting myotonic dystrophy (DM1). J. Am Chem Soc. 2008; 130: 16254-16261.

68. Jahromi AH, Nguyen L, Fu Y, Miller KA, Baranger AM, Zimmerman SC. A novel CUG(exp)·MBNL1 inhibitor with therapeutic potential for myotonic dystrophy type 1. ACS Chem Biol. 2013; 8: 1037-1043.

69. Childs-Disney JL, Hoskins J, Rzuczek SG, Thornton CA, Disney MD. Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive. ACS Chem Biol. 2012; 7: 856-862.

70. Kumar A, Parkesh R, Sznajder LJ, Childs-Disney JL, Sobczak K, Disney MD. Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r (CAG)- containing transcripts. ACS Chem Biol. 2012; 7: 496-505.

71. Tran T, Childs-Disney JL, Liu B, Guan L, Rzuczek S, Disney MD. Targeting the r (CGG) repeats that cause FXTAS with modularly assembled small molecules and oligonucleotides. ACS Chem Biol. 2014; 9: 904-912.

72. Su Z, Zhang Y, Gendron TF, Bauer PO, Chew J, Yang WY, et al. Discovery of a biomarker and lead small molecules to target r(GGGGCC)- associated defects in c9FTD/ALS. Neuron. 2014; 83: 1043-1050.

73. Pushechnikov A, Lee MM, Childs-Disney JL, Sobczak K, French JM, Thornton CA, et al. Rational design of ligands targeting triplet repeating transcripts that cause RNA dominant disease: application to myotonic muscular dystrophy type 1 and spinocerebellar ataxia type 3. J Am Chem Soc. 2009; 131: 9767-9779.

74. Velagapudi SP, Disney MD. Two-dimensional combinatorial screening enables the bottom-up design of a microRNA-10b inhibitor. Chem Commun (Camb). 2014; 50: 3027-3029.

75. Velagapudi SP, Gallo SM, Disney MD. Sequence-based design of bioactive small molecules that target precursor microRNAs. Nat Chem Biol. 2014; 10: 291-297.

76. Bose D, Jayaraj G, Suryawanshi H, Agarwala P, Pore SK, Banerjee R, et al. The tuberculosis drug streptomycin as a potential cancer therapeutic: inhibition of miR-21 function by directly targeting its precursor. Angew Chem Int Ed Engl. 2012; 51: 1019-1023.

77. Stephenson ML, Zamecnik PC. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A. 1978; 75: 285-288.

78. Zamecnik PC, Stephenson ML. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A. V; 75: 280-284.

79. Keller W, Crouch R. Degradation of DNA RNA hybrids by ribonuclease H and DNA polymerases of cellular and viral origin. Proc Natl Acad Sci U S A. 1972; 69: 3360-3364.

80. Agrawal S. Importance of nucleotide sequence and chemical modifications of antisense oligonucleotides. Biochim Biophys Acta. 1999; 1489: 53-68.

81. Arechavala-Gomeza V, Khoo B, Aartsma-Rus A. Splicing modulation therapy in the treatment of genetic diseases. Appl Clin Genet. 2014; 7: 245-252.

82. Farooqi AA, Rehman ZU, Muntane J. Antisense therapeutics in oncology: current status. Onco Targets Ther. 2014; 7: 2035-2042.

83. Lorenzer C, Dirin M, Winkler AM, Baumann V, Winkler J. Going beyond the liver: progress and challenges of targeted delivery of siRNA therapeutics. J Control Release. 2015; 203: 1-15.

84. Crooke ST. Molecular mechanisms of antisense drugs: RNase H. Antisense Nucleic Acid Drug Dev. 1998; 8: 133-134.

85. Wu H, Lima WF, Zhang H, Fan A, Sun H, Crooke ST. Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs. J Biol Chem. 2004; 279: 17181-17189.

86. Galarneau A, Min KL, Mangos MM, Damha MJ. Assay for evaluating ribonuclease H-mediated degradation of RNA-antisense oligonucleotide duplexes. Methods Mol Biol. 2005; 288: 65-80.

87. Stelzer AC, Kratz JD, Zhang Q, Al-Hashimi HM. RNA dynamics by design: biasing ensembles towards the ligand-bound state. Angew Chem Int Ed Engl. 2010; 49: 5731-5733.

88. Stelzer AC, Frank AT, Kratz JD, Swanson MD, Gonzalez-Hernandez MJ, Lee J, et al. Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble. Nat Chem Biol. 2011; 7: 553-559.