Open access peer-reviewed chapter

Non-Coding RNAs and Cancer

Written By

Gianpiero Di Leva and Michela Garofalo

Submitted: November 8th, 2011Reviewed: November 12th, 2012Published: January 24th, 2013

DOI: 10.5772/54997

Chapter metrics overview

2,762 Chapter Downloads

View Full Metrics

1. Introduction

The question of which regions of the human genome constitute its functional elements—those expressed as genes or serving as regulatory elements—has long been a central topic in biology. In the 1970s and 1980s, early cloning-based methods revealed the presence of more than 7000 genes in human genome [1], and large-scale analyses of expressed sequence tags (ESTs) in the 1990s suggested that the estimated number of human genes range from 35,000 to 100,000 [2]. The completion of the human genome project narrowed the focus considerably by highlighting the surprisingly small number of protein-coding genes, which is now conventionally cited as less than 25,000 [3]. While the number of protein-coding genes (20,000–25,000) has maintained broad consensus, recent studies of the human transcriptome have revealed an astounding number of non-coding RNAs (ncRNAs) [4-6]. In fact, the increased sensitivity of genome tiling arrays provides an even more detailed view, revealing that the extent of non-coding sequence transcription is at least four times greater than coding sequence, and that the abundance of non-coding transcripts had been previously overlooked. The RNA world hypothesis proposes that early life was based on RNAs, which subsequently devolved the storage of information to more stable DNA, and catalytic functions to more versatile proteins. Consequently, despite crucial roles in the ancient processes of translation and splicing, RNA is assumed to have been largely relegated to an intermediate between gene and protein, encapsulated in the central dogma ‘DNA makes RNA makes protein’ [7]. However, the finding that most of the genome in complex organisms is transcribed and the discovery of new classes of regulatory non-coding RNAs (ncRNAs) challenges this assumption and suggests that RNAs have continued to evolve and expand alongside proteins and DNA.

ncRNAs are considered as RNA transcripts that do not encode for a protein. In the past decade, a great diversity of ncRNAs has been observed. Depending on the type of ncRNA, transcription can occur by any of the three RNA polymerases (RNA Pol I, RNA Pol II, or RNA Pol III). General conventions divide ncRNAs into two main categories: small ncRNAs less than 200 bp and long ncRNAs greater than 200 bps [8]. Within these two categories, there are also many individual classes of ncRNAs (Table1), although the degree of biological and experimental support for each class ranges substantially and should be evaluated individually. The relevance of ncRNAs in gene regulation has been rapidly unveiling during the last decade. However, the functional elements in the primary sequence of noncoding genes that determine their role as RNA molecules remain unknown. Protein-coding genes have a defined language with a set of grammatical rules: three nucleotides forms a codon that translates into a specific amino acid [9]. Aberrations in codons of a protein-coding gene can be interpreted in terms of the amino acids they encode. We can recognize a mutation in a codon and determine its contribution to a given disease. In contrast to the genetic code for protein synthesis, ‘the ncRNA alphabet’ – a specific set of RNA sequences or structural motifs important for ncRNA function – remains to be largely elucidated. However, it has become increasingly apparent that the ncRNAs are of crucial functional importance for normal development, physiology and disease [10]. The functional relevance of the ncRNAs is particularly evident for a class of small non-coding RNAs called microRNAs (miRNAs) [11-12]. In human diseases, particularly cancer, it has been shown that epigenetic and genetic defects in miRNAs and their processing machinery are a common hallmark of disease [13-16]. However, miRNAs are just the tip of the iceberg, and other ncRNAs such as small nucleolar RNAs (snoRNAs), PIWI-interacting RNAs (piRNAs), large intergenic non-coding RNAs (lincRNAs) and, overall, the heterogeneous group of long non-coding RNAs (lncRNAs), might also contribute to the development of many different human disorders. Here we discuss the most recent genetic studies on ncRNAs and their related proteins in the context of cancer and we will analyze the new regulatory elements of the noncoding language to interpret their contribution to the pathogenesis of cancer.

Advertisement

2. MicroRNAs

In 1993, Victor Ambros and colleagues discovered a gene, lin-4, that affected development in Caenorhabditiselegansand found that its product was a small nonprotein-coding RNA [31]. The number of known small RNAs in different organisms such as Caenorhabditis elegans, Drosophila melanogaster, plants, and mammals—including humans—has since expanded substantially, mainly as a result of the cloning and sequencing of size-fractionated RNAs. MiRNAs are single stranded RNAs (ssRNAs) of 19–25 nucleotides in length that are generated from endogenous hairpin transcripts [32]. They play an important role in the negative regulation of gene expression by base-pairing to partially complementary sites on the target messenger RNAs (mRNAs), usually in the 3’ untranslated region (UTR). Binding of a miRNA to the target mRNA typically leads to translational repression and exonucleolytic mRNA decay, although highly complementary targets can be cleaved endonucleolytically. A genomic analysis of miRNAs has revealed that more than 50% of mammalian miRNAs are located within the intronic regions of annotated protein-coding or non-protein-coding genes [33]. These miRNAs could therefore use their host gene transcripts as carriers, although it remains possible that some are actually transcribed separately from internal promoters. Other miRNAs, located in intergenic regions, apparently have their own transcriptional regulatory elements and thus constitute

Table 1.

Non coding RNA in human genome.

independent transcription units. Animal miRNAs are processed from longer primary transcripts (pri-miRNAs) that can contain multiple miRNAs [34,35]. Few pri-miRNA transcripts have been studied in detail, but in general miRNAs are regulated and transcribed similar to protein encoding genes by (Pol) II with the exception of the rapidly evolving RNA polymerase (Pol) III transcribed miRNA cluster [36]. MiRNA processing occurs in three essential steps (Figure 1). First, the nuclear endoribonuclease protein Drosha recognizes the miRNA hairpins in the primary transcript and cleaves each hairpin ~11 nt from its base [37-38]. It has been proposed that Drosha may recognize the pri-miRNA through the stem-loop structure and then cleave the stem at a fixed distance from the loop to liberate the pre-miRNA. How is the Drosha enzyme able to discriminate the pri-miRNA stem-loop structure from the other stem-loop cellular RNAs? Both cell culture experiments and in vitro Drosha cleavage assays have shown that proteins associated with Drosha confer specificity to this process. In fact, Drosha has been found to be part of a large, ~650-kDa protein complex known as the Microprocessor [39], where Drosha interacts with its cofactor DGCR8 (the DiGeorge syndrome critical region gene 8 protein) in the human and interacts with Pasha in Drosophila melanogaster[40]. The next step in miRNA biogenesis is recognition of the ~60 nt pre-miRNA by exportin-5 and export into the cytoplasm in a ran-guanine-GTP-dependent manner [41-43]. The Exp5/Ran-GTP complex has a high affinity for pre-miRNAs,

Figure 1.

miRNA biogenesis and function. The primary miRNA (pri-miRNA) is transcribed by RNA pol II from its genomic location and cleaved by the microprocessor complex, which comprises Drosha and DGCR8. The resulting pre-miRNA is actively transported to the cytoplasm by exportin 5 (Expt.5), where the pre-miRNA undergoes further processing into the mature miRNA by Dicer and its co-factors, protein activator of interferon-induced protein kinase (PACT) and TAR RNA binding protein (TRBP). Normally, one strand of this duplex is degraded (miRNA star), whereas the other strand accumulates as a mature miRNA. From the miRNA-miRNA duplex, only the miRNA enters preferentially in the protein effector complex, formed by the RNA-induced silencing complex (RISC) and miRgonaute and binds with partial complementarity to the 3′ untranslated region (UTR) of target messenger RNAs (mRNAs) to mediate translational repression.

protecting them from the moment they are generated in the nucleus until they are ready for the next cleavage step in the cytoplasm, where GTP is hydrolyzed to guanosine diphosphate (GDP); at that point, the Exp5/Ran-GDP complex releases its cargo. Third, the endoribonuclease protein Dicer cleaves the pre-miRNA into ~22 nt duplexes and, with the help of cofactors such as TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT), preferentially incorporates one of the duplex strands Into the RNA induced-silencing complex (RISC) [44-50]. The final product is a miRNA-miRNA duplex that needs to be unwound to act as a single-stranded guide in the RISC to recognize its target mRNAs. It was originally proposed that an ATP-dependent helicase (known as unwindase) separates the two small RNA strands, after which the resulting single-stranded guide is loaded into Ago proteins. However, it was later shown that DrosophilaAgo2 [51], as well as human Ago2 [52], directly receive double-stranded small RNA from the RISC-loading complex. Ago2 then cleaves the passenger strand, thereby liberating the single-stranded guide to form mature Ago2-RISC. In mammals, miRNAs guide the RISC to complementary target sites in mRNAs, where endonucleolytically active Ago proteins cleave the RNA [53] (Figure 1). Finally, RISC can cleave [54-55] degrade [56-57] or suppress translation [58-59] of target mRNAs depending on the complementarity between miRNA and mRNA. Imperfect base pairing between small RNAs and their target mRNAs leads to repression of translation and/or deadenylation (removal of the polyA tail of the target), followed by destabilization of the target [60], whereas perfect base pairing usually leads to mRNA degradation.

Advertisement

2. MicroRNAs and cancer

Cancer is a multistep process in which normal cells experience genetic changes that progress them through a series of pre-malignant states (initiation) into invasive cancer (progression) that can spread throughout the body (metastasis). The dysregulation of genes involved in cell proliferation, differentiation and/or apoptosis is associated with cancer initiation and progression. Genes linked with cancer development are characterized as oncogenes and tumor suppressors. Recently, the definition of oncogenes and tumor suppressors has been expanded from the classical protein coding genes to include miRNAs [61-62]. MiRNAs have been found to regulate more than 60% of mRNAs and have roles in fundamental processes, such as development [63], differentiation [64], cell proliferation [65], apoptosis [66], and stress responses [67]. Over the past few years, many miRNAs have been implicated in various human cancers. The first evidence that miRNAs are involved in cancer comes from the finding that miR-15 and miR-16 are downregulated or deleted in most patients with chronic lymphocytic leukemia [68]. This discovery has projected miRNAs to the center stage of molecular oncology and, in the past few years, a myriad of genome-wide miRNA expression profiling analyses have shown a general dysregulation of miRNA expression in all tumors (Table 2) [69]. Surprisingly, the use of miRNA profiles is newly becoming highly preferred to the traditional mRNA signature for a variety of reasons. First, the remarkable stability of miRNAs, due to their short length, has allowed scientists to perform analyses also in samples considered to be technically challenging, such as formalin fixed specimens. High sensitive and refined miRNA detection technique provide high reliability in the use of miRNAs as a diagnostic tools. Finally, miRNA fingerprints have demonstrated the ability to identify the tissue of origin for cancer that have already spread in multiple metastatic sites, thereby reducing patient’s psychological burden and overall procedure costs. To date, over 1000 miRNAs have been reported in humans (miRbase: 1527 at November 2011), and both loss and gain of miRNA functions contribute to cancer development through a range of different mechanisms that we will discuss in the following sections.

a
b
c

Table 2.

miRNA profiling in cancer.

Advertisement

3. Oncogenic microRNAs

Although studies linking miRNA dysfunctions to human diseases are in their infancy, a great deal of data already exists, establishing an important role for miRNAs in the pathogenesis of cancer. Many miRNAs have been shown to function as oncogenes in the majority of cancers profiled to date (Table 3). MiR-21displays a strong evolutionary conservation across a wide range of vertebrate species in mammalian, avian and fish clades [70]. It has been demonstrated that a primary transcript containing miR-21(i.e., pri-miR-21) is independently transcribed from a conserved promoter that is located within the intron of the overlapping protein-coding gene TMEM49[71]. Several studies suggest that this miRNA is oncogenic [72-74] and that it may act as an antiapoptotic factor. For example, Chan et al. have found that miR-21 is commonly and markedly up-regulated in human glioblastoma and that inhibiting miR-21 expression leads to caspase activation and associated apoptotic cell death [72]. Moreover, Zhu and collaborators provided the first evidence that miR-21regulates invasion and metastasis, at least in part, by targeting metastasis-related tumor suppressor genes such as TPM1, programmed cell death 4 (PDCD4) and maspin [73]. Furthermore, examination of human breast tumor specimens revealed an inverse correlation of miR-21with PDCD4 and maspin [74]. The final proof of miR-21 oncogenic activity came from the Slack laboratory where the first conditional knock-in of miR-21 overexpressing mice was generated. The mice developed a severe pre-B-cell lymphoma but when miR-21 was reduced to endogenous levels, the mouse tumors completely disappeared, defining the concept of “oncomiR addition” [75].

Another important oncogenic miRNA is represented by miR-155. Several groups have shown that miR-155is highly expressed in pediatric Burkitt’s lymphoma [76], Hodgkin’s disease [77], primary mediastinal non-Hodgkin’s lymphoma [77], chronic lymphocytic leukemia (CLL) [78], acute myelogenous leukemia (AML) [79], lung cancer [80], pancreatic cancer [81], and breast cancer [80]. Dr. Croce laboratory reported that miR-155transgenic mice develop acute lymphoblastic leukemia/high-grade lymphoma and that most of these leukemias start at approximately nine months, irrespective of the mouse strain, preceded by a polyclonal pre-B-cell proliferation [82].

Another example of “oncomiR” is represented by miR-221&222cluster that is highly upregulated in a variety of solid tumors, including thyroid cancer [83], hepatocarcinoma [84], estrogen receptor negative breast tumor [85], and melanoma [86]. Elevated miR-221&222expression has been causally linked to proliferation [85-87], apoptosis [88-89], and migration [89] of several cancer cell lines. We recently reported that the hepatocyte growth factor receptor (MET) oncogene, through c-Jun transcriptional activation, upregulates miR-221&222expression, which, in turn, by targeting PTENand TIMP3, confers resistance to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and enhances tumorigenicity of lung and liver cancer cells [89]. The results suggest that therapeutic intervention involving the use of miRNAs should not only sensitize tumor cells to drug-inducing apoptosis but also inhibit their survival, proliferation, and invasion [89].

The miR-106b-25 polycistron is composed of the highly conserved miR-106b, miR-93, and miR-25 that accumulate in different types of cancer, including gastric, prostate, and pancreatic neuroendocrine tumors, as well as neuroblastoma and multiple myeloma. Petrocca and collaborators [90] demonstrated that E2F1 regulates miR-106b, miR-93, and miR-25, inducing their accumulation in gastric tumors. Conversely, miR-106b and miR-93 control E2F1 expression, establishing a negative feedback loop that may be important in preventing E2F1 self-activation and apoptosis. On the other hand, miR-106b, miR-93, and

Table 3.

-oncomiRs

miR-25 overexpression causes a decreased response of gastric cancer cells to TGFβ by downregulating p21 and Bim, the two most downstream effectors of TGFβ-dependent cell cycle arrest and apoptosis, respectively.

Another example of a miRNA locus with oncogenic properties is represented by the miR-17-92cluster, which consists of six miRNAs: miR-17-5p, -18, -19a, -19b, -20a, and -92-1. The miR-17-92 cluster is located in a region frequently amplified in several types of lymphoma and solid tumors [91-92]. It has been shown that mice deficient for miR-17-92 die shortly after birth with lung hypoplasia and a ventricular septal defect. This cluster is also essential for B cell development; its absence, in fact, leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B-to-pre-B transition [93]. All together these studies indicate that many miRNAs have oncogenic activity. Importantly, their knockdown through the use of antisense oligonucleotides, inhibits the development of cancer-associated phenotypes, laying the groundwork for the creation of miRNA-based therapies [94-96].

Advertisement

3. Tumor suppressor microRNAs

The first evidence that miRNAs are involved in cancer comes from the finding that miR-15 and miR-16 are downregulated or deleted in most patients with chronic lymphocytic leukemia (CLL) (Table 4) [68]. They are transcribed as a cluster (miR-15a–miR-16-1) that resides in the 13q14 chromosomal region. Deletions or point mutations in region 13q14 occur at high frequency in CLL, lymphoma, and several solid tumors [97]. Their expression is inversely correlated to BCL2expression in CLL [98]. The tumor suppressor function of miR-15a/16-1has also been addressed in vivo. In immunocompromised nude mice, ectopic expression of miR-15a/16-1was found to cause dramatic suppression of tumorigenicity of MEG-01 leukemic cells that exhibited a loss of endogenous expression of miR-15a/16-1. Furthermore, Klein et al. [99] generated transgenic mice with a deletion of the miR-15a–miR-16-1cluster, causing development of indolent B-cell-autonomous, clonal lymphoproliferative disorders, recapitulating the spectrum of CLL-associated phenotypes observed in humans. Recently, Bonci et al. reported that the miR-15a–miR-16-1cluster targets not only BCL2but also CCND1(encoding cyclin D1) and WNT3AmRNA, which promote several prostate tumorigenic features, including survival, proliferation, and invasion [100]. Together, these data suggest that miR-15a/16-1genes are natural antisense interactors of BCL2and probably other oncogenes and that they can be used to suppress tumor growth in therapeutic application for a variety of tumors [100].

In mammalians, the miR-34 family comprises three processed miRNAs that are encoded by two different genes: miR-34a is encoded by its own transcript, whereas miR-34b and miR-34c share a common primary transcript. The miR-34 family has been shown to form part of the p53 tumor-suppressor network: their expression is directly induced by p53 in response to DNA damage or oncogenic stress [101-102]. He et al. identified different miR-34 targets such as cyclin E2 (CCNE2), CDK4, and MET. Silencing these selected miR-34 targets through the use of small interfering RNAs (siRNAs) led to a substantial cell cycle arrest in G1. Moreover, ectopic miR-34 delivery caused a decrease in levels of phosphorylated retinoblastoma gene product (Rb), consistent with lowered activity of both CDK4 and CCNE2 complexes [102]. BCL2 and MYCN were also identified as miR-34a targets and likely mediators of the tumor suppressor phenotypic effect in neuroblastoma [103]. It has been also reported that p53 activation suppressed the EMT-inducing transcription factor SNAIL via induction of the miR-34a/b/c genes. In fact, suppression of miR-34a/b/c by anti-miRs caused up-regulation of SNAIL and cells displayed EMT markers, enhanced migration and invasion [104].

MicroRNA-122 (miR-122) is a liver-specific microRNA and is frequently downregulated in liver cancer [105]. Xu et al. reported that restoration of miR-122 in hepatocellular carcinoma cells could render cells sensitive to chemotherapeutic agents adriamycin or vincristine through downregulating antiapoptotic gene Bcl-w and cell cycle related gene cyclin B1 [106]. Another group found that over-expression of miR-122 inhibits hepatocellular carcinoma cell growth and promotes the cell apoptosis by affecting Wnt/β-catenin signalling pathway [107]. Coulouarn et al. showed that miR-122 is specifically repressed in a subset of primary hepatocellular tumors that are characterized by poor prognosis [108]. They further reported that loss of miR-122 resulted in an increase of cell migration and invasion and that restoration of miR-122 reverses this phenotype [108]. The final understanding of the tumor suppressor role for mir-122 role in liver cancer came from a recent study where miR-122 knockout mice were studied. When miR-122 KO mice aged, hepatic inflammation ensued, preceding the progressive onset of fibrosis and, eventually, tumors resembling human liver cancer. These pathologic manifestations were associated with hyperactivity of oncogenic pathways and hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokines, including IL-6 and TNF [109].

Table 4.

Tumor suppressor miRS

Advertisement

4. MetastamiRs

Metastasis is the result of cancer cells detaching from a primary tumor, consequently adapting to distant tissues and organs, and forming a secondary tumor [110] and this ability of cancer cells to metastasize is a hallmark of malignant tumors [111-112]. To successfully metastasize, a tumor cell must complete a complex set of processes, including invasion, survival and arrest in the circulatory system, and colonization of foreign organs. Despite great advancements in knowledge of metastasis biology, the molecular mechanisms are still not completely understood. Several miRNAs have been shown to initiate invasion and metastasis by targeting multiple proteins that are major players in these cellular events, thus they have been denominated as metastamiRs (Table 5). It seems that these metastasis-associated miRNAs do not influence primary tumor either in development or initiation steps of tumorigenesis, but they regulate key steps in the metastatic program and processes, such as epithelial-mesenchymal transition (EMT), apoptosis, and angiogenesis. Ma et. al reported that miR-10b is highly expressed in metastatic breast cancer cells and positively regulates cell migration and invasion. Overexpression of miR-10b in otherwise non-metastatic breast tumors initiates robust invasion and metastasis [113]. The team led by Joan Massague found that miR-335, miR-126, and miR-206 are metastasis-suppressors in breast cancer [114]. MiR-126 and miR-206 restoration reduced overall tumor growth and proliferation, whereas miR-335 inhibits metastatic cell invasion through targeting of the progenitor cell transcription factor SOX4and extracellular matrix component tenascin C [114]. Others miRNAs with prominent roles in breast cancer metastasis have been reported. It has been reported that miR-31 inhibited multiple steps of metastasis including invasion, anoikis, and colonization leading to almost complete reduction of lung metastasis [115]. Clinically, miR-31 levels were lower in breast cancer patients with metastasis. In addition, miR-9, which is up-regulated in breast cancer cells, directly targets CDH1, the E-cadherin-encoding messenger RNA, leading to increased cell motility and invasiveness [116].

Another important aspect of the metastatic dissemination is represented by the epithelial-to-mesenchymal transition (EMT) that allow neoplastic cells to abandon their primary site and survive in the new tissue. During EMT, an epithelial neoplastic cell looses cell adhesion by repressing E-cadherin expression and thereby the cell increases its motility. Numerous studies have shown that different microRNAs are modulated during EMT and one of the best-studied example is represented by the miR-200 family. These miRs are commonly lost in aggressive tumors such as lung, prostate, and pancreatic cancer. It has been shown that miR-200 family members directly target ZEB1 and ZEB2, transcription repressors of E-cadherin [117]. In fact, in the highly aggressive mouse lung cancer model where KRAS is constitutively activated and p53 function is perturbed, miR-200 ectopic expression prevented metastasis by repressing ZEB1 and ZEB2 and preventing E-cadherin down-regulation [117]. However, overexpression of the miR-200 family is associated with an increased risk of metastasis in breast cancer and this overexpression promotes metastatic colonization in mouse models, phenotypes that cannot be explained by E-cadherin expression alone [118]. By using proteomic profiling of the targets of mesenchymal-to-ephitelial (MET)-inducing miR-200, the authors discovered that miR-200 globally targets secreted proteins in breast cancer cells. Between the 38 modulated target genes, Sec23a, which is involved in transporting protein cargo from the endoplasmic reticulum to the Golgi, shows a superior association with human metastatic breast cancer as compared to the currently recognized miR-200 targets ZEB1 and the EMT marker E-cadherin. EMT is first acquired in the onset of transmigration and then reversed in the new metastatic site. Korpal et al. have shown that the miR-200 status predicts predisposition of the cancer to successful metastasis [119].

Table 5.

metastamiRS

Advertisement

5. Other non-coding RNAs: Biology and implications in cancer

5.1. snoRNAs: From post-transcriptional modification to cancer

Small nucleolar RNAs (snoRNAs) have, for many years, been considered one of the best-characterized classes of non-coding RNAs (ncRNAs) [120-123] but despite the common assumption that snoRNAs only have cellular housekeeping functions, in the past few years, independent reports have converged in implicating snoRNAs in the control of cell fate and oncogenesis [124-130]. SnoRNAs are small RNAs of 60-300nt in lenght that specifically accumulate in the nucleolar compartment of the cell where are in charge of the 2′-O-ribose methylation and pseudouridylation of specific ribosomal RNA nucleotides, essential modification for the efficient and accurate production of the ribosome [120-122]. The snoRNAs carry out their function in the form of small nucleolar ribonucleoproteins (snoRNPs), each of which consists of a box C/D or box H/ACA guide RNA, and four associated C/D or H/ACA snoRNP proteins (Figure 2). In both cases, snoRNAs hybridize specifically to the complementary sequence in the rRNAs, and the associated protein complexes then carry out the appropriate modification on the nucleotide that is identified by the snoRNAs. Biogenesis of vertebrate snoRNPs is remarkable and highly variable: in fact snoRNA gene organization ranges from independently transcribed genes, endowed with their own promoter elements, to intronic coding units lacking an independent promoter. In both yeast and animals, processing of intron-encoded snoRNAs is largely splicing-dependent; in contrast, the production of plant snoRNAs from introns seems to rely on a splicing-independent process [131]. Moreover, in both contexts (intergenic or intronic), genes can be either single or part of clusters. In the latter case, the generation of individual snoRNAs involves the enzymatic processing of polycistronic precursor RNAs. Such a processing, at least in yeast, appears to involve the same combination of endo- and exoribonucleases required for the maturation of monocistronic pre-snoRNAs [132-134]. The first indication that snoRNAs might have important roles in human disease was provided by the genetic studies on Prader–Willi syndrome (PWS), an inherited human disorder characterized by a complex phenotype, including mental retardation, decreased muscle tone and failure to thrive at birth, short stature, hypogonadism, sleep apnea, behavioral problems and hyperphagia (an insatiable appetite) that can lead to severe obesity [135]. The disease is caused by the genomic loss of the imprinted chromosomic 15q11-q13 locus which is normally only active on the paternal allele. The only characterized and conserved genes within this 121-kb-long genomic interval are the numerous HBII-85 snoRNA gene copies, thus suggesting that loss of expression of these repeated small C/D RNA genes might play a role in conferring some (or even all) phenotypes of the human disease and PWS-like phenotypes in mice (neonatal lethality, growth retardation and hypotonia). In fact, it has been shown that a site-specific deletion of the entire murine MBII-85 gene cluster led to post-natal growth retardation with low postnatal lethality (<15%) only seen in some genetic backgrounds, but no obesity [136]. Although all the imprinted C/D RNAs that have been tested accumulate within the nucleolus, none of them appear to act as RNA guides to modify rRNAs or spliceosomal U-snRNAs; they are called ‘orphan C/D RNAs’. So far, the MBII-52 gene clusters have attracted much attention, given that the neuronal-specific MBII-52 small RNA is predicted to interfere (A-to-I RNA editing and/or alternative RNA splicing) with the post-transcriptional regulation of the pre-mRNA that encodes the 5-HT2C (5-hydroxytryptamine 2C) receptor, playing a key role in regulating serotonergic signal transduction [137-138]. These observations raised the possibility that snoRNAs could have functions completely independent from their traditional activities and carry out other regulatory roles. The first insights into the potential roles of snoRNAs in cancer began with a study that identified C/D box snoRNA U50 and its host gene U50HG at the breakpoint in the t(3;6) (q21;q15) translocation in a diffuse large B cell lymphoma [139]. Moreover, snoRNAU50 gene has been found to undergo to a frequent copy number loss and a transcriptional downregulation in breast and prostate cancer samples [139,140]. In addition, a 2-bp deletion in U50 sequence also occurred both somatically and in germline, leading to increased incidence of homozygosity for the deletion in cancer cells [140].

Figure 2.

snoRNAs. A.Boxed sequences C and D (named from conserved, nuclease-resistant sequences that were originally identified in snoRNA U3) are hallmarks of the C/D box snoRNAs; boxed sequences H (Hinge region) and ACA are hallmarks of the H/ACA box snoRNAs. These conserved boxed sequences are important for the associations with protein components that are required to form the functional small nucleolar ribonucleoprotein (snoRNP) complexes and for accumulation in the nucleolus. C/D box snoRNAs associate with several proteins, including fibrillarin, which is the methyl transferase that is involved in the 2′-O-methylation of particular ribonucleotides, and H/ACA box snoRNAs associate with proteins such as the pseudouridine synthase dyskerin. Antisense sequences within the C/D box and H/ACA box snoRNAs guide the snoRNP complex to the appropriate nucleotide within the target RNA (most often ribosomal RNA). In a minority of cases both C/D-associated and C′D′-associated antisense sequences within the same C/D box snoRNA can act as guides for 2′-O-methylation of the target RNA. The eukaryotic H/ACA box snoRNAs contain two hairpin domains with complementary regions flanking the uridine to be converted in the target rRNA, at a position 14–16 nucleotides upstream of the conserved H and/or ACA box. Most mammalian snoRNAs are encoded within the introns of genes producing 5′ terminal oligopyrimidine (5′TOP) RNAs.B. Organization of snoRNA genes in representative eukaryotic genomesC. Small nucleolar RNAs (snoRNAs) in vertebrate are predominantly located in introns. Following splicing, debranching and trimming, mature snoRNAs are either exported, in which case they function in ribosomal RNA (rRNA) processing, or remain in the nucleus, where they are involved in alternative splicing and additional yet unknown functions.

SnoRNA42 (SNORA42) is located on chromosome 1q22 which is a commonly frequent amplified genomic region in lung cancer and overexpression of SNORA42 is frequently and remarkably found in NSCLC cells [141]. In addition, SNORA42 exhibited close correlations between its increases of copy number and expression level, suggesting that SNORA42 overexpression could be activated through its amplification. Importantly, engineered repression of SNORA42 caused marked repression of lung cancer growth in vitro and in vivo and it is associated with increased apoptosis by a p53-dependent pathway. Although not exhibiting apoptosis, p53 null and mutant p53 cancer cells with reduced levels of SNORA42 also show inhibited proliferation and growth, suggesting that SNORA42 knockdown can inhibit cell proliferation in p53-dependent or -independent manner. These independent studies on U50 and SNORA42 provide evidence for the functional importance of snoRNAs in cancer, and they show that snoRNAs can promote, as well as suppress, tumour development. In 2002, Wu and coworkers demonstrated that the expression of snoRNAs 5S was differentially displayed in different tissues and noticeably was highly expressed in normal brain, but its expression drastically decreased in meningioma [142]. Recently, genome-wide approaches identified six snoRNAs (SNORD33, SNORD66, SNORD73B, SNORD76, SNORD78, and SNORA42) that were statistically differently expressed between the non small cell lung cancer tumor and paired noncancerous samples [143]. Specifically, all these snoRNAs displayed a strong up-regulation in lung tumor specimens and the majority of them is located in commonly frequent genomic amplified regions in lung cancer: SNORD33 is located in chromosome 19q13.3 that contain potential oncogenes in lung cancer, while SNORD66 and SNORD76 are situated in chromosomal regions 3q27.1 and 1q25.1, respectively 3q27.1 and 1q25.1 are two of the most frequently amplified chromosomal segments in solid tumors, particularly NSCLC [143].

As well as the initial evidence that snoRNAs are involved in cancer development, there are some preliminary data showing that the genes that host snoRNAs might also contribute to the aetiology of this disease. A research screening for potential tumor-suppressor genes identified that Growth arrest-specific transcript 5 (gas5) gene as almost undetectable in actively growing cells but highly expressed in cells undergoing serum starvation or density arrest [144-145]. Gas5 is a multi-snoRNA host gene which encodes 9 (in mouse) or 10 (in human) snoRNAs and like all known snoRNA host genes exhibit characteristics which belong to the class of genes encoding 5′ terminal oligopyrimidine (5′TOP) mRNAs [146]. The first and stronger evidence that GAS5 is related to cancer is the identification that GAS5 transcript levels are significantly reduced in breast cancer samples relative to adjacent unaffected normal breast epithelial tissues and some, but not all, GAS5 transcripts sensitize mammalian cells to apoptosis inducers [147]. Other studies have also showed that GAS5 reduced expression is associated with poor prognosis in both breast cancer and head and neck squamous cell carcinoma [148]. Of note, GAS5 has been also identified as a novel partner of the BCL6 in a patient with diffuse large B-cell lymphoma, harboring the t(1;3)(q25;q27) [149]. Another example of a mature spliced transcript that harbors C/D-box snoRNAs and can function independently of the snoRNAs is represented by the transcript Zfas1 [150]. This gene intronically hosts three C/D box snoRNAs (Snord12, Snord12b, and Snord12c) and has been identified as one of the most differentially expressed gene during mouse mammary development. siRNA-mediated downregulation of Zfas1 mRNA in a mouse mammary cell line increased proliferation and differentiation without substantially affecting the levels of the snoRNA hosted within its intron. The human homologue, ZFAS1 (also known as ZNFX1‐AS1), which is predicted to share secondary structural features with mouse Zfas1, is expressed at high levels in the mammary gland and is downregulated in breast cancer. Taken together, these findings indicates that snoRNA host genes might have important functions in regulating cellular homeostasis and, potentially, cancer biology but more studies are needed to understand their involvement in molecular basis of disease and classify them as sources of potential biomarkers and therapeutic targets.

Another important aspect of the association between snoRNAs and tumorigenesis is represented by the involvement of their associated proteins in cancer. A point mutations in the DKC1 gene is the cause of a rare X-linked recessive disease, the dyskeratosis congenita (DC) [151-152]. Individuals with DC display features of premature aging, as well as nail dystrophy, mucosal leukoplakia, interstitial fibrosis of the lung, and increased susceptibility to cancer. DKC1 codes for dyskerin, a putative pseudouridine synthase, which carries out two separate functions, both fundamental for proliferating cells. One function is the pseudo-uridylation of ribosomal RNA (rRNA) molecules as a part of the H/ACA ribonucleoprotein complex, and the other is the stabilization of the telomerase RNA component necessary for telomerase activity. Dkc1 mutant mice recapitulate the major features of DC, including an increased susceptibility to tumor formation. Early generation (G1 and G2) of Dkc1 mutant mice showed a full spectrum of DC and presented alterations in rRNA modification, whereas defects in telomere length were not evident until G4 mice, suggesting that deregulated ribosome function is important for the initiation of DC and that impairment in telomerase activity in Dkc1 mutant mice may modify and/or exacerbate the disease in later generations. To this regard, DKC1 was identified as one of only seventy genes that, collectively, constitute a gene expression profile that strongly correlates with the development of aneuploidy and is associated with poor clinical prognosis in a variety of human cancers. Therefore, one hypothesis is that an alteration of physiologic dyskerin function, irrespective of the mechanism, may perturb mitosis and contribute to tumorigenesis but this idea will require more detailed investigation. Another possibility is related to the strong effect of dyskerin loss on H/ACA accumulation. Recent finding in fact have shown that some H/ACA box and C/D box can be processed to produce small RNAs, at least some of which can function like miRNAs [153]. Such processing may be of crucial importance, as miRNAs have important roles in the development of many cancers as previously discussed. To date, Xiao and colleagues have recently reported that an H/ACA box snoRNA- derived miRNA, miR-605, has a key role in stress-induced stabilization of the p53 tumour suppressor protein [154]. p53 transcriptionally activates its negative regulator, MDM2, in addition to miR-605. miR-605 counteracts MDM2 through post-transcriptional repression; under conditions of stress, this snoRNA-derived miRNA offsets the MDM2 negative-feedback loop, generating a positive-feedback loop to enable the rapid accumulation of p53. However, whether this regulation of p53 by miR-605 is relevant to cancer biology has not yet been addressed. Like dyskerin, NHP2 and NOP10 proteins, both components of the H/ACA snoRNPs, are also significantly up-regulated in sporadic cancers and high levels may be associated with poor clinical prognosis. Moreover, germline NHP2 and NOP10 mutations give rise to autosomal recessive forms of dyskeratosis congenita, and cancer susceptibility is also a feature of these genetic forms of the disease. Since the functions of several snoRNAs have not yet been identified (orphan snoRNAs), it is possible that disruption of snoRNP biogenesis by any mechanism may affect an array of important cellular processes, and could potentiate cancer development and/or progression.

5.2. piRNAs: Guardians of the genome

Piwi-interacting RNAs (piRNAs) are germline-specific small silencing RNAs of 24–30 nt in length, that suppress transposable elements (TE) activity and maintain genome integrity during germline development, a role highly conserved across animal species [155-156]. TEs are genomic parasites that threaten the genomic integrity of the host genome: they are able to move to new sites by insertion or transposition and thereby disrupt genes and alter the genome [157]. In animals, endogenous siRNAs also silence TEs, but the piRNA pathway is at the forefront of defense against transposons in germ cells [158]. piRNAs specifically associate with PIWI proteins, which are germline-specific members of the AGO protein family, AGO3, Aubergine (Aub) and Piwi, and form a piRNA-induced silencing complex (piRISC) which will guide the TE silencing [159-162]. Any mutations in each of the three members of the PIWI family lead to transposon derepression in the germline, indicating that they act non-redundantly during TE silencing. Initial screening of piRNA sequences revealed that there are hundreds of thousands, if not millions, of individual piRNA sequences [163-165]. Furthermore, they are characterized by the absence of specific sequence motifs or secondary structures such as miRNA precursors. Despite their large diversity, most piRNAs can be mapped to a relatively small number of genomic regions called piRNA clusters. Each cluster extends from several to more than 200 kilobases, it contains multiple sequences that generate piRNAs and some piRNAs map to both genomic strands, suggesting bidirectional transcription [163-165] Indeed, analysis of piRNA clusters in different Drosophilaspecies has shown that, although the clusters locations are conserved, their sequence content has evolved very quickly suggesting adjustments in the piRNAs patrimony in order to suppress new active transposons invading the species. Therefore, piRNA clusters may be considered as repositories of information, enabling production of many mature piRNAs that target diverse TEs. Two main pathways, highly conserved in many animal species, have been discovered to be responsible for the biogenesis of the piRNAs: the primary pathways and the Ping-Pong amplification (Figure 3) [166-168]. First, the primary piRNA biogenesis pathway provides an initial pool of piRNAs that target multiple TEs. Next, the Ping-Pong cycle further shapes the piRNA population by amplifying sequences that target active transposons. It is currently unclear how primary piRNAs are produced from piRNA clusters but it is likely that piRNA precursors are single-stranded and therefore do not require Dicer for their processing. Interestingly, piRNAs that associate with each member of the PIWI protein family have a distinct size,suggesting that PIWI proteins can act as ‘rulers’ that define the size of mature piRNAs. Several additional proteins (e.s. Zucchini, Armitage and Yb) have also been identified that are involved in primary piRNA biogenesis and mutations in and/or depletion of any of these three proteins eliminates primary piRNAs associated with PIWI proteins. In some cell types, such as somatic follicle cells of the D. melanogasterovary, primary piRNA biogenesis is the only mechanism that generates piRNAs. However, in germline cells of the D. melanogasterovary and in the pre-meiotic spermatogonia in mice, there is another mechanism called the Ping-Pong cycle that amplifies specific sequences generated by the primary biogenesis pathway [163,169]. Mainly the Ping-Pong pathway engages AGO3 and Aubergine, both of which are accumulated in perinuclear structures located at the cytoplasmic face of the nuclear envelope in animal germline cells, named “nuage”. The pathway depends on the endoribonuclease or Slicer activity of AGO3 and Aubergine, which act catalytically one after the other, leading to a cleavage of the target RNAs between their tenth and eleventh nucleotides relative to the ‘guide’ small RNAs. This process results in the generation of repeated rounds of piRNA production having exactly the same sequence of the original primary piRNA. The ping-pong pathway amplifies piRNAs in D. melanogastertestes, especially those originating from TEs. Non-TE-derived piRNAs seem to be barely amplified by the amplification loop. This two steps of piRNA biogenesis can be compared with the function of the adaptive immune system in protecting against pathogens. The primary piRNA biogenesis pathway resembles the initial generation of the hypervariable antibody repertoire, whereas the amplification loop is analogous to antigen-directed clonal expansion of antibody-producing lymphocytes during the acute immune response. An emerging number of studies highlight the role of piRNAs or PIWI proteins in the regulation of tumorigenesis. First examples of the piRNA involvement in cancer is represented by the up-regulation of HIWI, one of the four human Piwi homologues, in about 60 % of seminomas [170]. In fact, HIWI maps to a locus known as a germ cell tumor susceptibility locus (12aq24.33). HIWI overexpression has also been found in somatic cells such as soft-tissue sarcomas or ductal pancreas adenocarcinoma, and strongly correlates with bad prognosis and high incidence of tumor-related death, providing an example for a potential tumorigenic role of a piRNA-related protein in somatic cells [171,172]. In some cancers, PIWIL2 overexpression has been suggested to induced resistance in cells to cisplatin, which might arise because of increased chromatin condensation that prevents the normal process of DNA repair [173]. Furthermore, new high-throughput sequencing data revealed the presence of piRNAs in somotic cells, such as HeLa cells. These somatic piRNAs appear located in the nucleolus and in the cytoplasmic area surrounding the nuclear envolope and in contrast with the large population of known piRNAs in male germ cells, this population of piRNAs is dramatically smaller [174]. Another recent study demonstrated that the level of piR-651 is significantly higher in several cancer histotype including lung, mesothelium, breast, liver, and cervical cancer compared to non-cancerous adjacent tissues and inhibition of piR-651 induced block of gastric cancer cells at the G2/M phase [175,176]. Another example is represented by the downregulation of piR-823 in gastric cancer tissues; its enforced expression inhibited gastric cancer cell growth in vitro and in vivo, suggesting a tumor suppressive properties for piR-823 [177]. Interestingly, piRNAs not are only involved in direct regulation by degradation of TE but they have also been linked to DNA methylation of the retrotraspon regions, extending piRNA functions beyond post-transcriptional silencing. In fact, CpG DNA methylation, which is required for efficient transcriptional silencing of LINE and LTR retrotransposons in the genome, is decreased in the male germ line of mice with defective PIWI proteins. Specifically, mice with defective PIWI proteins fail to establish de novo methylation of TE sequences during spermatogenesis, leading to the hypothesis that the piRISC can also guide the de novomethylation machinery to TE loci. In this scenerio, piRNAs may present a perfect guide for discriminating TE sequences from normal protein-coding genes and marking them for DNA methylation; however, the biochemical details of how these two mechanisms of piRNA action might be linked have not yet been revealed [178,179]. All together, these data revealed that PIWI-associated RNAs and PIWI pathway has a more profound function outside germline cells than was originally thought but many more studies are needed to clarify their specific role in tumorigenesis.

Figure 3.

piRNAs. A, schematic representation of the Drosophila egg chamber.B,piRNAs (which are 24–32 nt in length) are processed from single-stranded RNA precursors that are transcribed largely from mono- or bidirectional intergenic repetitive elements known as piRNA clusters. Unlike miRNAs and siRNAs, piRNAs do not require Dicer for their processing. First, primary piRNAs are produced through the primary processing pathway and are amplified through the ping-pong pathway, which requires Slicer activity of PIWI proteins. Subsequently, additional piRNAs are produced through a PIWI-protein-catalysed amplification loop (called the 'ping-pong cycle') via sense and antisense intermediates. Primary piRNA processing and loading onto mouse PIWI proteins might occur in the cytoplasm. The PIWI ribonucleoprotein (piRISC) complex functions in transposon repression through target degradation and epigenetic silencing.C, total number of piRNA clusters in different animal species according to the piRNA Database (http://pirnabank.ibab.ac.in/).

Advertisement

6. The emergence of long non-coding RNAs

Over the last decade, advances in genome-wide analyses of the eukaryotic transcriptome have revealed that most of the human genome is transcribed, generating a large repertoire of (>200 nt) long non-coding RNAs (lncRNA or lincRNA, for long intergenic ncRNA) that map to intronic and intergenic regions [181,181]. Given their unexpected abundance, lncRNAs were initially thought to be spurious transcriptional noise resulting from low RNA polymerase fidelity [182]. However, the restricted expression of many long ncRNAs to particular developmental contexts, the often exhibiting precise subcellular localization and the binding of transcription factors to non-coding loci, suggested that a significant portion of ncRNAs fulfills functional roles beyond transcriptional remodelling [183-187]. lncRNA typically refers to a polyadenylated long ncRNA that is transcribed by RNA polymerase II and is associated with epigenetic signatures common to protein-coding genes, such as trimethylation of histone 3 lysine 4 (H3K4me3) at the transcriptional start site (TSS) and trimethylation of histone 3 lysine 36 (H3K36me3) throughout the gene body [188-189]. lncRNAs also commonly exhibit splicing of multiple exons into a mature transcript, and their transcription occurs from an independent gene promoter and is not coupled to the transcription of a nearby or associated parental gene. RNA-Seq studies now suggest that several thousand uncharacterized lncRNAs are present in any given cell type [188-189], and that the human genome may harbor nearly as many lncRNAs as protein-coding genes (perhaps ~15,000 lncRNAs), although only a fraction is expressed in a given cell type. One main characteristic of the lncRNAs is their very low sequence conservation that had fueled the idea that they are not functional. This assertion needs to be carefully considered and takes in consideration several points. First, a recent study identified the presence of 1,600 lncRNAs that show a strong evolutionary conservation and function ranging from from embryonic stem cell pluripotency to cell proliferation [189]. In contrast to the protein coding genes, long ncRNAs can exhibit shorter stretches of sequence that are conserved to maintain functional domains and structures. Indeed, many long ncRNAs with a known function, such as Xist, only exhibit high conservation over short sections of their length [190]. Third, rather than being indicative of non-functionality, low sequence conservation can also be explained by high rates of primary sequence evolution if long ncRNAs have, like promoters and other regulatory elements, more plastic structure–function constraints than proteins [190]. The diverse selection pressures acting on long ncRNAs probably reflect the wide range of their functions which can be regrouped in three major subclasses: chromatin remodeling, transcriptional modulation and nuclear architecture/subnuclear localization.

long ncRNAs can mediate epigenetic changes by recruiting chromatin remodelling complexes to specific genomic loci resolving the paradox of how a small repertoire of chromatin remodelling complexes are able To specify the large array of chromatin modifications without any apparent specificity for the genomic loci [191,192]. A recent study found that 20% of 3300 human long non coding RNAs are bound by Polycomb Repressive Complex 2 (PRC2) [193]. Although the specific molecular mechanisms are not defined, there are several examples that can illustrate the silencing potential of lncRNAs (Figure 4). The first most known example is represented by the X-chromosome inactivation which is carried out by a number of lncRNAs including Xist and RepA, which bind PRC2 complex, and the antogonist of Xist, Tsix [194]. In pre-X-inactivation cells, Tsix competes with RepA for the binding of PRC2 complex; when the X-inactivation starts Tsix is downregulated and PRC2 becomes available to RepA which can actively induced the transcription of Xist. The up-regulated Xist in turn preferentially binds to PRC2 and spreads across the chromosome X inducing PCR2-mediated trimethylated histone H3 lysine27. Another important example is represented by the hundreds of long ncRNAs which are sequentially expressed along the temporal and spatial developmental axes of the human homeobox (Hox) loci, where they define chromatin domains of differential histone methylation and RNA polymerase accessibility [195]. One of these ncRNAs, Hox transcript antisense RNA (HOTAIR), originates from the HOXC locus and silences transcription across 40 kb of the HOXD locus in trans by inducing a repressive chromatin state, which is proposed to occur by recruitment of the Polycomb chromatin remodelling complex PRC2 by HOTAIR (Figure 4). Recently, it has been proposed that HOTAIR has the ability to bind other histone-modifying enzymes such as the demethylase LSD1 [196]. In fact, knockdown of HOTAIR induces a rapid loss of LSD1 or PRC2 at hundreds of gene loci with the corresponding increase in expression. This model fits other chromatin modifying complexes, such as Mll, PcG, and G9a methyltransferase, which can be similarly directed by their associated ncRNAs [196]. As modulator of epigenetic landmark, it has been shown that HOTAIR has a profound effect on tumorigenesis. In fact, HOTAIR is upregulated in breast carcinoma and colon cancer and its correlates with metastasis and poor prognosis [197] Enforced expression of HOTAIR consistently changed the pattern of occupancy of Polycomb proteins from the typical epithelial mammary cells pattern to that of embryonic fibroblasts [198]. Another important effect of lncRNAs on chromatin modification that can highlight their impact on cancer is the relationship between the lncRNA ANRIL and the INK4b/ARF/INK4a locus, encoding for three tumor-suppressor genes highly deleted or silenced in a large cohort of tumors [199]. ANRIL, which is transcribed antisense to the protein coding genes of the locus, controls the epigenetic status of the locus by interacting with subunits of PRC1 and PRC2. High expression of ANRIL is found in some cancer tissues and is associated to a high levels of PCR-mediated trimethylated histone H3 lysine27. Inhibition of ANRIL releases PRC1 and PRC2 complexes from the locus, decreases the histone methylation status with the following increase of the protein coding gene transcription. Many other tumor suppressor genes that are frequently silenced by epigenetic mechanisms in cancer also have antisense partners, which can affect gene expression with different other mechanism. First, antisense ncRNAs can mask key cis-elements in mRNA by the formation of RNA duplexes, as in the case of the Zeb2 antisense RNA, which complements the 5′ splice site of an intron of Zeb2 mRNA [200]. Expression of the ncRNA prevents the splicing of the intron that contains an internal ribosome entry site required for efficient translation and expression of the ZEB2 protein with a further efficient translation (Figure 4). In this context, it has been evaluated that the prevalence of lncRNAs are antisense to introns, hypothesizing their role in the regulation of splicing or capable of generating mRNA duplexes that fuel the RISC machinery to silence gene expression. One major emergent theme is the involvement of the lncRNAs in the assembly or activity of transcription factors functioning as a scaffold for the docking of many proteins, mimicking functional DNA elements or modulation of PolII itself. The first example is represented by the suppression of CCND1 mediated by the lncRNAs through the recruitment and integration of the RNA binding protein TLS into a transcriptional programme. DNA damage signals induce the expression of long ncRNAs associated with the cyclin D1 gene promoter, where they act cooperatively to recruit the RNA binding

Figure 4.

lncRNAs.Schematic representation of the control operated on protein coding gene by the lncRNAs at the level of chromatin remodelling, transcriptional control and post-transcriptional processing.A, lncRNAs (Xist, HOTAIR, ANRIL, etc) can recruit chromatin modifying complexes to specific genomic loci to localize their catalytic activity. In this case, the lncRNA recruits the Polycomb complex by inducing trimethylation of the lysine 27 residues (me3K27) of histone H3 to produce heterochromatin formation and repress gene expression.B,C,D, lncRNAs can regulate the transcriptional process through a range of mechanisms. First, lncRNAs tethered to the promoter of the cyclin D1 gene recruit the RNA binding protein TLS to modulate the histone acetyltransferase activity of CREB binding protein (CBP) and p300 to repress gene transcription. Second, an ultraconserved enhancer is transcribed as a long ncRNA, Evf2, which subsequently acts as a co-activator to the transcription factor DLX2, to regulate the Dlx6 gene transcription. Third, a lncRNA transcribed from the DHFR minor promoter form a triplex at the major promoter to reduce the access of the general transcription factor TFIID, and thereby suppress DHFR gene expression.E, a lncRNA is antisense to Zeb2 mRNA and mask the 5′ splice site resulting in intron retention. This retention results in an efficient Zeb2 translation related to the presence of an internal ribosome entry site (IRE) in the retained intron.

protein TLS. The modified and promoter-docked TLS inhibits the histone acetyltransferase activities of CReB binding protein and p300 inducing the silencing of cyclin D1 expression (Figure 4) [201]. A different co-activator activity mediated by lncRNAs is also evident in the regulation of Dlx genes, important modulators of neuronal development and patterning [202]. Dlx5-6 expression is regulated by two ultraconserved enhancers one of which is transcribed in a lncRNA, named Evf-2. Evf2 forms a stable complex with the homodomein protein DLX-2 which in turn acts as a transcriptional enhancer of Dxl5-6 gene (Figure 4). In some cases, lncRNAs can also affect RNA polymerase activity by influencing the initiation complex in the choice of the promoter. For example, in humans, a ncRNA transcribed from an upstream region of the dihydrofolate reductase (DHFR) locus forms a triplex in the major promoter of DHFR to prevent the binding of the transcriptional co-factor TFIID (Figure 4). This could be a widespread mechanism for controlling promoter usage as thousands of triplex structures exist in eukaryotic chromosomes. Recently, lncRNAs have also shown their tumorigenic potential by modulating the transcriptional program of p53 [203]. An 3kb lncRNAs, linc-RNA-p21, transcriptionally activated by p53, has been shown to collaborate with p53 in order to control the gene expression in response to DNA damage. Specifically, silencing of lincRNA-p21 derepresses the expression of hundred of genes which are also derepressed following p53 knockdown. It has also been discovered that lincRNA-p21 interacts with hnRNPK and this binding is essential for the modulation of p53 activity.

The final category of lncRNAs is represented by those molecules capable to generate the formation of compartmentalized nuclear organelles, subnuclear membraneless nuclear bodies whose funtion is relative unknown. One of them is represented by cell-cycle regulated nuclear foci, named paraspeckles. In addition to protein components, two lncRNAs, NEAT1 and Men epsilon, have been detected as essential part of the paraspeckles. While depletion of NEAT or Men epsilon disrupts the paraspeckles, their overexpression strongly increases their number. There is a number of different lncRNAs that localize to different nuclear regions [204]. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) localizes to the splicing speckles, Xist and Kcnq1ot1 both, localize to the perinucleolar region during the S phase of the cell cycle, a class of repeat-associated lncRNAs (es SatIII) are associated to nuclear stress bodies which are produced on specifc pericentromeric heterochromatic domains containing SatIII gene itself.

Advertisement

7. Conclusions

Alterations in microRNAs and other short or long non-coding RNA (ncRNA) are involved in the initiation, progression, and metastasis of human cancer. Over the last decade, a growing number of non-coding transcripts have been found to have roles in gene regulation and RNA processing. The most well known small non-coding RNAs are the microRNAs, but the network of long and short non-coding transcripts is complex and is likely to contain as yet unidentified classes of molecules that form transcriptional regulatory networks. The field of small and long non coding RNAs is rapidly advancing toward in vivo delivery for therapeutic purposes. Advanced molecular therapies aimed at downmodulating or upmodulating the level of a given miRNA in model organisms have been successfully established. RNA-based gene therapy can be used to treat cancer by using RNA or DNA molecules as therapy against the mRNA of genes involved in cancer pathogenesis or by directly targeting the ncRNAs that participate in pathogenesis. The use of miRNAs is still being evaluated preclinically; no clinical or toxicologic studies have been published but the future is promising. Kota and collegues reported that systemic administration of this miRNA in a mouse model of HCC using adeno-associated virus (AAV) results in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity (116). Recently, Pineau et al. (117) identified DNA damage-inducible transcript 4 (DDIT4), a modulator of the mTor pathway, as a bona fide target of miR-221. They introduced into liver cancer cells, by lipofection, LNA-modified oligonucleotides specifically designed for miR-221 (antimiR-221) and miR-222 (antimiR-222) knockdown. Treatment by antagomiRs, but not scrambled oligonucleotide, reduced cell growth in liver cancer cell lines that overexpressed miR-221 and miR-222 by 35% and 22%, respectively. Thus the use of synthetic inhibitors of miR-221 may prove to be a promising approach to liver cancer treatment (117). Despite recent progress in silencing of miRNAs in rodents, the development of effective and safe approaches for sequence-specific antagonism of miRNAs in vivo remains a significant scientific and therapeutic challenge. Recently, Elmen and collaborators (118) showed for the first time, that the simple systemic delivery of an unconjugated, PBS-formulated LNA-antimiR effectively antagonizes the liver-expressed miR-122 in nonhuman primates. Administration by intravenous injections of LNA-antimiR into African green monkeys resulted in the formation of stable heteroduplexes between the LNA-antimiR and miR-122, accompanied by depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. These findings demonstrate the utility of systemically administered LNA-antimiRs in exploring miRNA functions in primates and show the impressive potential of this strategy to overcome a major hurdle for clinical miRNA therapy. In conclusion, the discovery of small RNAs and their functions has revitalized the prospect of controlling expression of specific genes in vivo, with the ultimate hope of building a new class of gene-specific medical therapies. Just how significant are the ncRNAs? They appear to be doing something important and highly sophisticated; there are so many of them, their sequences are so highly conserved, their expression is tissue specific, and they have recognition sites on more than 30% of the entire transcriptome. It seems that ncRNAs were overlooked in the past simply because researchers were specifically looking for RNAs that code proteins. The above discussed data highlight that the complexity of genomic control operated by the ncRNAs is somewhat greater than previously imagined, and that they could represent a total new order of genomic control. In this scenario, understanding the precise roles of ncRNAs is a key challenge. The targeting of other ncRNAs, in addition to miRNAs, is still in its infancy, but new important developments are expected in this area. Therefore, small RNAs could become powerful therapeutic tools in the near future.

References

  1. 1.MatsubaraKOkuboK1993Identification of new genes by systematic analysis of cDNAs and database construction.Curr Opin Biotechnol.4672677
  2. 2.LiangFHoltIPerteaGKaramychevaSSalzbergS. LQuackenbushJ2000Gene index analysis of the human genome estimates approximately 120,000 genes.Nat Genet.25239240
  3. 3.LanderE. SLintonL. MBirrenBNusbaumCZodyM. CBaldwinJDevonKDewarKDoyleMFitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, Doucette-Stamm L, Rubenfield M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Federspiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Raymond C, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la Bastide M, Dedhia N, Blöcker H, Hornischer K, Nordsiek G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Jang W, Johnson LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, Smit AF, Stupka E, Szustakowski J, Thierry-Mieg D, Thierry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, Morgan MJ, de Jong P, Catanese JJ, Osoegawa K, Shizuya H, Choi S, Chen YJ. (2001International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature.409860921
  4. 4.GuttmanMAmitIGarberMFrenchCLinM. FFeldserDHuarteMZukOCareyB. WCassadyJ. PCabiliM. NJaenischRMikkelsenT. SJacksTHacohenNBernsteinB. EKellisMRegevARinnJ. LLanderE. S2009Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammalsNature458223227
  5. 5.GuttmanMGarberMLevinJ. ZDonagheyJRobinsonJAdiconisXFanLKoziolM. JGnirkeANusbaumCRinnJ. LLanderE. SRegevA2010Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAsNat. Biotechnol.28503510
  6. 6.MarquesA. CPontingC. P2009Catalogues of mammalian long noncoding RNAs: modest conservation and incompletenessGenome Biol.10:R124.
  7. 7.CechT. R2009Crawling out of the RNA worldCell136599602
  8. 8.KapranovPChengJDikeSNixD. ADuttaguptaRWillinghamA. TStadlerP. FHertelJHackermüllerJHofackerI. LBellICheungEDrenkowJDumaisEPatelSHeltGGaneshMGhoshSPiccolboniASementchenkoVTammanaHGingerasT. R2007RNA maps reveal new RNA classes and a possible function for pervasive transcription.Science.31614841488
  9. 9.CrickF. HBarnettLBrennerSWatts-tobinR. J1961General nature of the genetic code for proteinsNature.19212271232
  10. 10.MercerT. RDingerM. EMattickJ. S2009Long non-coding RNAs insight into functions.Nature Rev. Genet.10155159
  11. 11.HeLHannonG. J2004MicroRNAs: small RNAs with a big role in gene regulation.Nature Rev. Genet.5522531
  12. 12.MendellJ. T2005MicroRNAs: critical regulators of development, cellular physiology and malignancy.Cell Cycle411791184
  13. 13.Esquela-kerscherASlackF. J2006OncomiRs- microRNAs with a role in cancer. Nature Rev. Cancer6259269
  14. 14.HammondS. M2007MicroRNAs as tumor suppressors.Nature Genet.39582583
  15. 15.CroceC. M2009Causes and consequences of microRNA dysregulation in cancer.Nature Rev. Genet.10704714
  16. 16.NicolosoM SSpizzoRShimizuMRossiSCalinG. A2009MicroRNAs- the micro steering wheel of tumour metastases. Nature Rev. Cancer.9293302
  17. 17.CarninciPKasukawaTKatayamaSGoughJFrithM. CMaedaNOyamaRRavasiTLenhardBWellsCKodziusRShimokawaKBajicV. BBrennerS. EBatalovSForrestA. RZavolanMDavisM. JWilmingL. GAidinisVAllenJ. EAmbesi-impiombatoAApweilerRAturaliyaR. NBaileyT. LBansalMBaxterLBeiselK. WBersanoTBonoHChalkA. MChiuK. PChoudharyVChristoffelsAClutterbuckD. RCroweM. LDallaEDalrympleB. PDe BonoBDella Gatta G, di Bernardo D, Down T, Engstrom P, Fagiolini M, Faulkner G, Fletcher CF, Fukushima T, Furuno M, Futaki S, Gariboldi M, Georgii-Hemming P, Gingeras TR, Gojobori T, Green RE, Gustincich S, Harbers M, Hayashi Y, Hensch TK, Hirokawa N, Hill D, Huminiecki L, Iacono M, Ikeo K, Iwama A, Ishikawa T, Jakt M, Kanapin A, Katoh M, Kawasawa Y, Kelso J, Kitamura H, Kitano H, Kollias G, Krishnan SP, Kruger A, Kummerfeld SK, Kurochkin IV, Lareau LF, Lazarevic D, Lipovich L, Liu J, Liuni S, McWilliam S, Madan Babu M, Madera M, Marchionni L, Matsuda H, Matsuzawa S, Miki H, Mignone F, Miyake S, Morris K, Mottagui-Tabar S, Mulder N, Nakano N, Nakauchi H, Ng P, Nilsson R, Nishiguchi S, Nishikawa S, Nori F, Ohara O, Okazaki Y, Orlando V, Pang KC, Pavan WJ, Pavesi G, Pesole G, Petrovsky N, Piazza S, Reed J, Reid JF, Ring BZ, Ringwald M, Rost B, Ruan Y, Salzberg SL, Sandelin A, Schneider C, Schönbach C, Sekiguchi K, Semple CA, Seno S, Sessa L, Sheng Y, Shibata Y, Shimada H, Shimada K, Silva D, Sinclair B, Sperling S, Stupka E, Sugiura K, Sultana R, Takenaka Y, Taki K, Tammoja K, Tan SL, Tang S, Taylor MS, Tegner J, Teichmann SA, Ueda HR, van Nimwegen E, Verardo R, Wei CL, Yagi K, Yamanishi H, Zabarovsky E, Zhu S, Zimmer A, Hide W, Bult C, Grimmond SM, Teasdale RD, Liu ET, Brusic V, Quackenbush J, Wahlestedt C, Mattick JS, Hume DA, Kai C, Sasaki D, Tomaru Y, Fukuda S, Kanamori-Katayama M, Suzuki M, Aoki J, Arakawa T, Iida J, Imamura K, Itoh M, Kato T, Kawaji H, Kawagashira N, Kawashima T, Kojima M, Kondo S, Konno H, Nakano K, Ninomiya N, Nishio T, Okada M, Plessy C, Shibata K, Shiraki T, Suzuki S, Tagami M, Waki K, Watahiki A, Okamura-Oho Y, Suzuki H, Kawai J, Hayashizaki Y; FANTOM Consortium; RIKEN Genome Exploration Research Group and Genome Science Group (Genome Network Project Core Group). (2005The transcriptional landscape of the mammalian genome. Science.30915591563
  18. 18.GibbE. ABrownC. JLamW. L2011The functional role of long non-coding RNA in human carcinomas.Mol Cancer 10:38 EOF
  19. 19.TaftR. JPangK. CMercerT. RDingerMMattickJ. S2010Non-coding RNAs: regulators of disease. J Pathol.22012639
  20. 20.Di Leva GCroce CM. (2010Roles of small RNAs in tumor formationTrends Mol Med.1625767
  21. 21.GarofaloMCroceC. M2011microRNAs: Master regulators as potential therapeutics in cancer.Annu Rev Pharmacol Toxicol.512543
  22. 22.ENCODE Project ConsortiumBirney E, Stamatoyannopoulos JA, Dutta A, Guigó R, Gingeras TR, Margulies EH, Weng Z, Snyder M, Dermitzakis ET, Thurman RE, Kuehn MS, Taylor CM, Neph S, Koch CM, Asthana S, Malhotra A, Adzhubei I, Greenbaum JA, Andrews RM, Flicek P, Boyle PJ, Cao H, Carter NP, Clelland GK, Davis S, Day N, Dhami P, Dillon SC, Dorschner MO, Fiegler H, Giresi PG, Goldy J, Hawrylycz M, Haydock A, Humbert R, James KD, Johnson BE, Johnson EM, Frum TT, Rosenzweig ER, Karnani N, Lee K, Lefebvre GC, Navas PA, Neri F, Parker SC, Sabo PJ, Sandstrom R, Shafer A, Vetrie D, Weaver M, Wilcox S, Yu M, Collins FS, Dekker J, Lieb JD, Tullius TD, Crawford GE, Sunyaev S, Noble WS, Dunham I, Denoeud F, Reymond A, Kapranov P, Rozowsky J, Zheng D, Castelo R, Frankish A, Harrow J, Ghosh S, Sandelin A, Hofacker IL, Baertsch R, Keefe D, Dike S, Cheng J, Hirsch HA, Sekinger EA, Lagarde J, Abril JF, Shahab A, Flamm C, Fried C, Hackermüller J, Hertel J, Lindemeyer M, Missal K, Tanzer A, Washietl S, Korbel J, Emanuelsson O, Pedersen JS, Holroyd N, Taylor R, Swarbreck D, Matthews N, Dickson MC, Thomas DJ, Weirauch MT, Gilbert J, Drenkow J, Bell I, Zhao X, Srinivasan KG, Sung WK, Ooi HS, Chiu KP, Foissac S, Alioto T, Brent M, Pachter L, Tress ML, Valencia A, Choo SW, Choo CY, Ucla C, Manzano C, Wyss C, Cheung E, Clark TG, Brown JB, Ganesh M, Patel S, Tammana H, Chrast J, Henrichsen CN, Kai C, Kawai J, Nagalakshmi U, Wu J, Lian Z, Lian J, Newburger P, Zhang X, Bickel P, Mattick JS, Carninci P, Hayashizaki Y, Weissman S, Hubbard T, Myers RM, Rogers J, Stadler PF, Lowe TM, Wei CL, Ruan Y, Struhl K, Gerstein M, Antonarakis SE, Fu Y, Green ED, Karaöz U, Siepel A, Taylor J, Liefer LA, Wetterstrand KA, Good PJ, Feingold EA, Guyer MS, Cooper GM, Asimenos G, Dewey CN, Hou M, Nikolaev S, Montoya-Burgos JI, Löytynoja A, Whelan S, Pardi F, Massingham T, Huang H, Zhang NR, Holmes I, Mullikin JC, Ureta-Vidal A, Paten B, Seringhaus M, Church D, Rosenbloom K, Kent WJ, Stone EA; NISC Comparative Sequencing Program; Baylor College of Medicine Human Genome Sequencing Center; Washington University Genome Sequencing Center; Broad Institute; Children’s Hospital Oakland Research Institute, Batzoglou S, Goldman N, Hardison RC, Haussler D, Miller W, Sidow A, Trinklein ND, Zhang ZD, Barrera L, Stuart R, King DC, Ameur A, Enroth S, Bieda MC, Kim J, Bhinge AA, Jiang N, Liu J, Yao F, Vega VB, Lee CW, Ng P, Shahab A, Yang A, Moqtaderi Z, Zhu Z, Xu X, Squazzo S, Oberley MJ, Inman D, Singer MA, Richmond TA, Munn KJ, Rada-Iglesias A, Wallerman O, Komorowski J, Fowler JC, Couttet P, Bruce AW, Dovey OM, Ellis PD, Langford CF, Nix DA, Euskirchen G, Hartman S, Urban AE, Kraus P, Van Calcar S, Heintzman N, Kim TH, Wang K, Qu C, Hon G, Luna R, Glass CK, Rosenfeld MG, Aldred SF, Cooper SJ, Halees A, Lin JM, Shulha HP, Zhang X, Xu M, Haidar JN, Yu Y, Ruan Y, Iyer VR, Green RD, Wadelius C, Farnham PJ, Ren B, Harte RA, Hinrichs AS, Trumbower H, Clawson H, Hillman-Jackson J, Zweig AS, Smith K, Thakkapallayil A, Barber G, Kuhn RM, Karolchik D, Armengol L, Bird CP, de Bakker PI, Kern AD, Lopez-Bigas N, Martin JD, Stranger BE, Woodroffe A, Davydov E, Dimas A, Eyras E, Hallgrímsdóttir IB, Huppert J, Zody MC, Abecasis GR, Estivill X, Bouffard GG, Guan X, Hansen NF, Idol JR, Maduro VV, Maskeri B, McDowell JC, Park M, Thomas PJ, Young AC, Blakesley RW, Muzny DM, Sodergren E, Wheeler DA, Worley KC, Jiang H, Weinstock GM, Gibbs RA, Graves T, Fulton R, Mardis ER, Wilson RK, Clamp M, Cuff J, Gnerre S, Jaffe DB, Chang JL, Lindblad-Toh K, Lander ES, Koriabine M, Nefedov M, Osoegawa K, Yoshinaga Y, Zhu B, de Jong PJ. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. (2007Nature.447799816
  23. 23.SiomiM. CSatoKPezicDAravinA. A2011PIWI-interacting small RNAs: the vanguard of genome defenceNat Rev Mol Cell Biol.1224658
  24. 24.FaulknerG. JKimuraYDaubC. OWaniSPlessyCIrvineK. MSchroderKCloonanNSteptoeA. LLassmannTWakiKHornigNArakawaTTakahashiHKawaiJForrestA. RSuzukiHHayashizakiYHumeD. AOrlandoVGrimmondS. MCarninciP2009The regulated retrotransposon transcriptome of mammalian cellsNat Genet.4156371
  25. 25.HuarteMRinnJ. L2010Large non-coding RNAs: missing links in cancer? Hum Mol Genet. 19:R15261
  26. 26.PauliARinnJ. LSchierA. F2011Non-coding RNAs as regulators of embryogenesisNat Rev Genet.1213649
  27. 27.CalinG. ALiuC. GFerracinMHyslopTSpizzoRSevignaniCFabbriMCimminoALeeE. JWojcikS. EShimizuMTiliERossiSTaccioliCPichiorriFLiuXZupoSHerleaVGramantieriLLanzaGAlderHRassentiLVoliniaSSchmittgenT. DKippsT. JNegriniMCroceC. M2007Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas.Cancer Cell1221529
  28. 28.PolisenoLSalmenaLZhangJCarverBHavemanW. JPandolfiP. P2010ACoding-independentfunction of gene and pseudogene mRNAs regulates tumour biology.Nature46510338
  29. 29.HeYVogelsteinBVelculescuV. EPapadopoulosNKinzlerK. W2008The antisense transcriptomes of human cellsScience32218557
  30. 30.KimT. KHembergMGrayJ. MCostaA. MBearD. MWuJHarminD. ALaptewiczMBarbara-haleyKKuerstenSMarkenscoff-papadimitriouEKuhlDBitoHWorleyP. FKreimanGGreenbergM. E2010Widespread transcription at neuronal activity-regulated enhancersNature4651827
  31. 31.LeeR. CFeinbaumR. LAmbrosV1993TheCelegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell.7584354
  32. 32.KimV. N2005MicroRNA biogenesis: coordinated cropping and dicing. Nat. Rev. Mol. Cell Biol.637685
  33. 33.RodriguezAGriffiths-jonesSAshurstJ. LBradleyA2004Identification of mammalian microRNA host genes and transcription units. Genome Res.14190210
  34. 34.Lagos-quintanaMRauhutRYalcinAMeyerJLendeckelWTuschlT2002Identification of tissue-specific microRNAs from mouse. Curr Biol.12735739
  35. 35.AltuviaYLandgrafPLithwickGElefantNPfefferSAravinABrownsteinM. JTuschlTMargalitH2005Clustering and conservation patterns of human microRNAs. Nucleic Acids Res3326972706
  36. 36.BorchertG. MLanierWDavidsonB. L2006RNA polymerase III transcribes human microRNAs. Nat Struct Mol Biol.1310971101
  37. 37.HanJLeeYYeomK. HNamJ. WHeoIRheeJ. KSohnS. YChoYZhangB. TKimV. N2006Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell125887901
  38. 38.SaetromPSnoveONedlandMGrunfeldT. BLinYBassM. BCannonJ. R2006Conserved microRNA characteristics in mammals. Oligonucleotides.1611544
  39. 39.GregoryR. IYanK. PAmuthanGChendrimadaTDoratotajBCoochNShiekhattarR2004The Microprocessor complex mediates the genesis of microRNAs. Nature43223540
  40. 40.HanJLeeYYeomK. HKimY. KJinHKimV. N2004The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev.18301627
  41. 41.YiRQinYMacaraI. GCullenB. R2003Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev.1730113016
  42. 42.LundEGuttingerSCaladoADahlbergJ. EKutayU2004Nuclear export of microRNA precursors. Science.3039598
  43. 43.BohnsackM. TCzaplinskiKGorlichD2004Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA.10185191
  44. 44.ManiatakiEMourelatosZ2005AHumanATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev.1929792990
  45. 45.LeeYHurIParkS. YKimY. KSuhM. RKimV. N2006The role of PACT in the RNA silencing pathway. EMBO J.25522532
  46. 46.HaaseA. DJaskiewiczLZhangHLaineSSackRGatignolAFilipowiczW2005TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep6961967
  47. 47.GregoryR. IChendrimadaT. PCoochNShiekhattarR2005Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell.123631640
  48. 48.ForstemannKTomariYDuTVaginV. VDenliA. MBratuD. PKlattenhoffCTheurkaufW. EZamoreP. D2005Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol. 3:e236.
  49. 49.ChendrimadaT. PGregoryR. IKumaraswamyENormanJCoochNNishikuraKNishikuraKShiekhattarR2005TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature.436740744
  50. 50.CastanottoDSakuraiKLingemanRLiHShivelyLAagaardLSoiferHGatignolARiggsARossiJ. J2007Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC. Nucleic Acids Res.35515464
  51. 51.RandT. APetersenSDuFWangX2005Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell.12362129
  52. 52.LeuschnerP. JAmeresS. LKuengSMartinezJ2006Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep.731420
  53. 53.MartinezJ,PatkaniowskaA,UrlaubH, Luhrmann R, Tuschl T. (2002). Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell. 110:563-74.
  54. 54.ZamoreP. DTuschlTSharpP. ABartelD. P2000RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell.1012533
  55. 55.YektaSShihI. HBartelD. P2004MicroRNA-directed cleavage of HOXB8 mRNA. Science.304594596
  56. 56.WuLFanJBelascoJ. G2006MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci USA.10340344039
  57. 57.BaggaSBrachtJHunterSMassirerKHoltzJEachusRPasquinelliA. E2005Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell.122553563
  58. 58.ReinhartB. JSlackF. JBassonMPasquinelliA. EBettingerJ. CRougvieA. EHorvitzH. RRuvkunG2000The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature403901906
  59. 59.OlsenP. HAmbrosV1999The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev Biol.216671680
  60. 60.PillaiR. SBhattacharyyaS. NFilipowiczW2007Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol.1711826
  61. 61.GarzonRFabbriMCimminoACalinG. ACroceC. M2006MicroRNA expression and function in cancer. Trends Mol. Med.12580587
  62. 62.WuWSunMZouG. MChenJ2007MicroRNA and cancer: Current status and prospective. Int. J. Cancer.120953960
  63. 63.BoettgerTBraunT2012A New Level of Complexity: The Role of MicroRNAs in Cardiovascular Development. Circ Res.11010001013
  64. 64.HanR,KanQ,SunY,WangS,ZhangG,PengT,JiaY. (2012).MiR-9 promotes the neural differentiation of mouse bone marrow mesenchymal stem cells via targeting zinc finger protein 521. Neurosci Lett. Ahead of print.
  65. 65.DUrsoP. I, DUrsoO. FStorelliCMallardoMGianfredaC. DMontinaroACimminoAPietroCMarsiglianteS2012MiR-155 is up-regulated in primary and secondary glioblastoma and promotes tumour growth by inhibiting GABA receptors. Int J Oncol.doi:ijo.2012.1420.
  66. 66.GarofaloMRomanoGDi Leva G, Nuovo G, Jeon YJ, Ngankeu A, Sun J, Lovat F, Alder H, Condorelli G, Engelman JA, Ono M, Rho JK, Cascione L, Volinia S, Nephew KP, Croce CM. (2011EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers. Nat Med.187482
  67. 67.FrankDGantenbergJBoomgaardenIKuhnCWillRJarrK. UEdenMKramerKLueddeMMairbäurlHKatusH. AFreyN2012MicroRNA-20a inhibits stress-induced cardiomyocyte apoptosis involving its novel target Egln3/PHD3. J Mol Cell Cardiol.52711717
  68. 68.CalinGA,DumitruCD, ShimizuM,BichiR, Zupo S, Noch E, Aldler H, Rattan S, Keating M, Rai K, Rassenti L, Kipps T, Negrini M, Bullrich F, Croce CM. (2002). Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA. 99:15524-29
  69. 69.CalinG. ACroceC. M2006MicroRNA signatures in human cancers. Nat Rev Cancer.6857866
  70. 70.CaiXHagedornC. HCullenB. R2004Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA.1019571966
  71. 71.CaiXHagedornC. HCullenB. R2004Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA.1019571966
  72. 72.ChanJ. AKrichevskyA. MKosikK. S2005MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res.6560296033
  73. 73.ZhuSWuHWuFNieDShengSMoY. Y2008MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res.18350359
  74. 74.ZhuQ,WangZ,HuY,LiJ,LiX,ZhouL,HuangY. (2012).miR-21 promotes migration and invasion by the miR-21-PDCD4-AP-1 feedback loop in human hepatocellular carcinoma. Oncol Rep.27:1660-1668.
  75. 75.MedinaP. PNoldeMSlackF. J2010OncomiR addiction in an in vivo model of microRNA-21-induced pre-B-cell lymphoma. Nature.4678690
  76. 76.MetzlerMWildaMBuschKViehmannSBorkhardtA2004High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma. Genes Chromosom. Cancer.39167169
  77. 77.KluiverJPoppemaSDe JongDBlokzijlTHarmsGJacobsSKroesenB. Jvan den Berg A. (2005BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas. J. Pathol.207243249
  78. 78.CalinG. AFerracinMCimminoADi Leva G, Shimizu M, Wojcik SE, Iorio MV, Visone R, Sever NI, Fabbri M, Iuliano R, Palumbo T, Pichiorri F, Roldo C, Garzon R, Sevignani C, Rassenti L, Alder H, Volinia S, Liu CG, Kipps TJ, Negrini M, Croce CM. (2005A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N. Engl. J. Med.35317931801
  79. 79.GarzonRVoliniaSLiuC. GFernandez-cymeringCPalumboTPichiorriFFabbriMCoombesKAlderHNakamuraTFlomenbergNMarcucciGCalinG. AKornblauS. MKantarjianHBloomfieldC. DAndreeffMCroceC. M2008MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia. Blood.11131833189
  80. 80.VoliniaSCalinG. ALiuC. GAmbsSCimminoAPetroccaFVisoneRIorioMRoldoCFerracinMPrueittR. LYanaiharaNLanzaGScarpaAVecchioneANegriniMHarrisC. CCroceC. M2006A microRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl. Acad. Sci. USA.10322572261
  81. 81.GreitherTGrocholaL. FUdelnowALautenschlagerCWUrlPTaubertH. (2010Elevated expression of microRNAs 155, 203, 210 and 222 in pancreatic tumors is associated with poorer survival. Int. J. Cancer.1267380
  82. 82.CostineanSZanesiNPekarskyYTiliEVoliniaSHeeremaNCroceC. M2006Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad Sci U S A.10370247029
  83. 83.PallantePVisoneRFerracinMFerraroABerlingieriM. TTronconeGChiappettaGLiuC. GSantoroMNegriniMCroceC. MFuscoA2006MicroRNA deregulation in human thyroid papillary carcinomas. Endocr. Relat. Cancer.13497508
  84. 84.FornariF,GramantieriL,FerracinM,VeroneseA,SabbioniS,CalinGA,GraziGL,GiovanniniC,CroceCM,BolondiL,NegriniM. (2008).MiR-221 control CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma. Oncogene. 27:5651-5661.
  85. 85.Di Leva GGasparini P, Piovan C, Ngankeu A, Garofalo M, Taccioli C, Iorio MV, Li M, Volinia S, Alder H, Nakamura T, Nuovo G, Liu Y, Nephew KP, Croce CM. (2010MicroRNA cluster 221-222 and estrogen receptor αinteractions in breast cancer. J. Natl. Cancer Inst.102706721
  86. 86.FelicettiFErricoM. CBotteroLSegnaliniPStoppacciaroABiffoniMFelliNMattiaGPetriniMColomboM. PPeschleCCareA2008The promyelocytic leukemia zinc finger-microRNA-221/-222 pathway controls melanoma progression through multiple oncogenic mechanisms. Cancer Res.6827452754
  87. 87.le Sage CNagel R, Egan DA, Schrier M, Mesman E, Mangiola A, Anile C, Maira G, Mercatelli N, Ciafrè SA, Farace MG, Agami R. (2007Regulation of the27Kip1tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation. EMBO J. 26:3699-3708.
  88. 88.GarofaloMQuintavalleCDi Leva G, Zanca C, Romano G, Taccioli C, Liu CG, Croce CM, Condorelli G. (2008MicroRNA signatures of TRAIL resistance in human non-small cell lung cancer. Oncogene.2738453855
  89. 89.GarofaloMDi Leva G, Romano G, Nuovo G, Suh SS, Ngankeu A, Taccioli C, Pichiorri F, Alder H, Secchiero P, Gasparini P, Gonelli A, Costinean S, Acunzo M, Condorelli G, Croce CM. (2009miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation. Cancer Cell.16498509
  90. 90.PetroccaFVisoneROnelliM. RShahM. HNicolosoM. SDe MartinoIIliopoulosDPilozziELiuC. GNegriniMCavazziniLVoliniaSAlderHRucoL. PBaldassarreGCroceC. MVecchioneA2008E2F1-regulated microRNAs impair TGFβ-dependent cell-cycle arrest and apoptosis in gastric cancer. Cancer Cell.13272286
  91. 91.OtaATagawaHKarnanSTsuzukiSKarpasAKiraSYoshidaYSetoM2004Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma. Cancer Res.6430873095
  92. 92.HayashitaYOsadaHTatematsuYYamadaHYanagisawaKTomidaSYatabeYKawaharaKSekidoYTakahashiT2005A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res.6596289632
  93. 93.VenturaAYoungA. GWinslowM. MLintaultLMeissnerAErkelandS. JNewmanJBronsonR. TCrowleyDStoneJ. RJaenischRSharpP. AJacksT2008Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell.132875886
  94. 94.ParkJ. KLeeE. JEsauCSchmittgenT. D2009Antisense inhibition of microRNA-21 or-221 arrests cell cycle, induces apoptosis, and sensitizes the effects of gemcitabine in pancreatic adenocarcinoma. Pancreas. 38:e190199
  95. 95.InomataMTagawaHGuoY. MKameokaYTakahashiNSawadaK2009MicroRNA-17-92 downregulates expression of distinct targets in different B-cell lymphoma subtypes. Blood.113396402
  96. 96.WangP. YLiY. JZhangSLiZ. LYueZXieNXieS. Y2010RegulatingAcells growth by ASO inhibiting miRNA expression. Mol. Cell. Biochem.339163171
  97. 97.BandiN,ZbindenS,GuggerM,ArnoldM,KocherV,HasanL,KappelerA,BrunnerT,VassellaE. (2009).miR-15a and miR-16 are implicated in cell cycle regulation in a Rb-dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer. Cancer Res. 69:5553-5559.
  98. 98.CimminoA,CalinGA,FabbriM,IorioMV,FerracinM,ShimizuM,WojcikSE,AqeilanRI,ZupoS,DonoM,RassentiL,AlderH,VoliniaS,LiuCG,KippsTJ,NegriniM,CroceCM. (2005).miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc. Natl. Acad. Sci. USA.102:13944-13949.
  99. 99.KleinULiaMCrespoMSiegelRShenQMoTAmbesi-impiombatoACalifanoAMigliazzaABhagatGDalla-faveraR2010The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer Cell172840
  100. 100.BonciD,CoppolaV,MusumeciM,AddarioA,GiuffridaR,MemeoL,DUrsoL,PagliucaA,BiffoniM,LabbayeC,BartucciM,MutoG,PeschleC,De MariaR. (2008).ThemiR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nat. Med. 14:1271-1277.
  101. 101.HeXHeLHannonG. J2007The guardian’s little helper: microRNAs in the53tumor suppressor network. Cancer Res.67:11099-11101.
  102. 102.HeLHeXLimL. PDe StanchinaEXuanZLiangYXueWZenderLMagnusJRidzonDJacksonA. LLinsleyP. SChenCLoweS. WClearyM. AHannonG. J2007A microRNA component of the53tumour suppressor network. Nature. 447:1130-1134.
  103. 103.ColeK. AAttiyehE. FMosseY. PLaquagliaM. JDiskinS. JBrodeurG. MMarisJ. M2008A functional screen identifies miR-34a as a candidate neuroblastoma tumor suppressor gene. Mol. Cancer Res.6735742
  104. 104.SiemensH,JackstadtR,HüntenS,KallerM,MenssenA,GötzU,HermekingH. (2011).miR-34 and SNAIL form a double-negative feedback loop to regulate epithelial-mesenchymal transitions. Cell Cycle.10:4256-4271.
  105. 105.TsaiW. CHsuP. WLaiT. CChauG. YLinC. WChenC. MLinC. DLiaoY. LWangJ. LChauY. PHsuM. THsiaoMHuangH. DTsouA. P2009MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma. Hepatology.4915711582
  106. 106.XuYXiaFMaLShanJShenJYangZLiuJCuiYBianXBiePQianC2011MicroRNA-122 sensitizes HCC cancer cells to adriamycin and vincristine through modulating expression of MDR and inducing cell cycle arrest. Cancer Lett.310160169
  107. 107.XuJZhuXWuLYangRYangZWangQWuF2012MicroRNA-122 suppresses cell proliferation and induces cell apoptosis in hepatocellular carcinoma by directly targeting Wnt/β-catenin pathway. Liver Int.32752760
  108. 108.CoulouarnCFactorV. MAndersenJ. BDurkinM. EThorgeirssonS. S2009Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Oncogene.2835263536
  109. 109.HsuS. HWangBKotaJYuJCostineanSKutayHYuLBaiSLa Perle K, Chivukula RR, Mao H, Wei M, Clark KR, Mendell JR, Caligiuri MA, Jacob ST, Mendell JT, Ghoshal K. (2012Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J Clin Invest.12228712883
  110. 110.NguyenD. XBosP. DMassagueJ2009Metastasis: From dissemination to organ-specific colonization. Nat. Rev. Cancer.9274284
  111. 111.HanahanDWeinbergR. A2000The hallmarks of cancer. Cell.1005770
  112. 112.SteegP. S2006Tumor metastasis: Mechanistic insights and clinical challenges. Nat. Med.12895904
  113. 113.MaLReinhardtFPanESoutschekJBhatBMarcussonE. GTeruya-feldsteinJBellG. WWeinbergR. A2010Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model. Nat. Biotechnol.28341347
  114. 114.TavazoieS. FAlarconCOskarssonTPaduaDWangQBosP. DGeraldW. LMassaguéJ2008Endogenous human microRNAs that suppress breast cancer metastasis. Nature.45114752
  115. 115.ValastyanSReinhardtFBenaichNCalogriasDSzászA. MWangZ. CBrockJ. ERichardsonA. LWeinbergR. A2009A pleiotropically acting microRNA, miR-31, inhibits breast cancer metastasis. Cell.13710321046
  116. 116.MaL,YoungJ,PrabhalaH,PanE,MestdaghP,MuthD,Teruya-FeldsteinJ,ReinhardtF,OnderTT,ValastyanS,WestermannF,SpelemanF,VandesompeleJ,WeinbergRA. (2010).MiR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat. Cell Biol.12: 247-256.
  117. 117.GregoryP. Aet alThe miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol,2008593601
  118. 118.GibbonsD. Let alContextual extracellular cues promote tumor cell EMT and metastasis by regulating miR-200 family expression. Genes Dev,200921402151
  119. 119.KorpalMet alDirect targeting of Sec23a by miR-200s influences cancer cell secretome and promotes metastatic colonization. Nat Med,201111011108
  120. 120.Kiss‐LaszloZ., Henry, Y., Bachellerie, J., Caizergues‐ Ferrer, M. & Kiss, T. (1996Site‐specific ribose methylation of preribosomal RNA: a novel function for small nucleolar RNAs. Cell.8510771088
  121. 121.TollerveyDKissT1997Function and synthesis of small nucleolar RNAs. Curr. Opin. Cell Biol.9337342
  122. 122.WeinsteinL. BSteitzJ. A1999Guided tours: from precursor snoRNA to functional snoRNP. Curr. Opin. Cell Biol.11378384
  123. 123.WilliamsG. THughesJ. PStonemanVAndersonC. LMccarthyN. JMourtada-maarabouniMPickardMHedgeV. LTraynerIFarzanehF2006Isolation of genes controlling apoptosis through their effects on cell survival. Gene Ther. Mol. Biol.10B:255261
  124. 124.Mourtada‐Maarabouni MHedge VL, Kirkham L, Farzaneh F, Williams GT. (2008Growth arrest in human T‐cells is controlled by the non‐coding RNA growth‐arrest‐specific transcript 5 (GAS5). J. Cell Sci.121939946
  125. 125.Mourtada‐Maarabouni MPickard MR, Hedge VL, Farzaneh F, Williams GT. (2009GAS5, a non‐protein‐coding RNA, controls apoptosis and is downregulated in breast cancer. Oncogene.28195208
  126. 126.DongX. YGuoPBoydJSunXLiQZhouWDongJ. T2009Implication of snoRNA U50 in human breast cancer. J. Genet. Genomics36447454
  127. 127.DongX. YRodriguezCGuoPSunXTalbotJ. TZhouWPetrosJLiQVessellaR. LKibelA. SStevensV. LCalleE. EDongJ. T2008SnoRNA U50 is a candidate tumor‐ suppressor gene at 6q14.3 with a mutation associated with clinically significant prostate cancer. Hum. Mol. Genet.1710311042
  128. 128.GeeH. EBuffaF. MCampsCRamachandranALeekRTaylorMPatilMSheldonHBettsGHomerJWestCRagoussisJHarrisA. L2011The small‐nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis. Br. J. Cancer.10411681177
  129. 129.LiaoJYuLMeiYGuarneraMShenJLiRLiuZJiangF2010Small nucleolar RNA signatures as biomarkers for non‐small‐cell lung cancer. Mol. Cancer. 9:198.
  130. 130.Martens-uzunovaE. SJalavaS. EDitsN. FVan LeendersG. JMollerSTrapmanJBangmaC. HLitmanTVisakorpiTJensterG2011Diagnostic and prognostic signatures from the small non‐coding RNA transcriptome in prostate cancer. Oncogene.31978991
  131. 131.BrownJ. WMarshallD. FEcheverriaM2008Intronic noncoding RNAs and splicing. Trends Plant Sci.13335342
  132. 132.ChanfreauGLegrainPJacquierA1998Yeast RNase III as a key processing enzyme in small nucleolar RNAs metabolism. J. Mol. Biol.284975988
  133. 133.PetfalskiEDandekarTHenryYTollerveyD1998Processing of the precursors to small nucleolar RNAs and rRNAs requires common components. Mol. Cell. Biol.1811811189
  134. 134.QuL. HHenrasALuY. LZhouHZhouW. XZhuY. QZhaoJHenryYCaizergues-ferrerMBachellerieJ. P1999Seven novel methylation guide small nucleolar RNAs are processed from a common polycistronic transcript by Rat1p and RNase III in yeast Mol. Cell. Biol.1911441158
  135. 135.NichollsR. DKnepperJ. L2001Genome organization, function, and imprinting in Prader-Willi and Angelman syndromes Annu. Rev. Genomics Hum. Genet.2153175
  136. 136.SkryabinB. VGubarL. VSeegerBPfeifferJHandelSRobeckTKarpovaERozhdestvenskyT. SBrosiusJ2007Deletion of the MBII-85 snoRNA gene cluster in mice results in postnatal growth retardation. PLoS Genet. 3:e235.
  137. 137.GurevichIEnglanderM. TAdlersbergMSiegalN. BSchmaussC2002Modulation of serotonin 2C receptor editing by sustained changes in serotonergic neurotransmission J. Neurosci.22105291053
  138. 138.BurnsC. MChuHRueterS. MHutchinsonL. KCantonHSanders-bushEEmesonR. B1997Regulation of serotonin-2C receptor G-protein coupling by RNA editing Nature.387303308
  139. 139.TanakaRSatohHMoriyamaMSatohKMorishitaYYoshidaSWatanabeTNakamuraYMoriS2000IntronicUsmall-nucleolar-RNA (snoRNA) host gene of no protein-coding potential is mapped at the chromosome breakpoint t(3;6) (q27;q15) of human B-cell lymphoma. Genes Cells.5277287
  140. 140.DongX. YRodriguezCGuoPSunXTalbotJ. TZhouWPetrosWLiQVessellaR. LKibelA. SStevensV. LCalleE. EDongD. J2008SnoRNA U50 is a candidate tumor-suppressor gene at 6q14.3 with a mutation associated with clinically significant prostate cancer Hum. Mol. Genet.1710311042
  141. 141.MeiY. PLiaoJ. PShenJ. PYuLLiuB. LLiuLLiR. YJiLDorseyS. GJiangZ. RKatzR. LWangJ. YJiangF2011Small nucleolar RNA 42 acts as an oncogene in lung tumorigenesis. Oncogene. (doi:10.1038/onc.2011.449).
  142. 142.ChangL. SLinS YLieuA. SWuT. L2002Differential expression of human 5S snoRNA genes. Biochem. Biophys. Res. Commun.299196200
  143. 143.LiaoJYuLMeiYGuarneraMShenJ2010Small nucleolar RNA signatures as biomarkers for non-small-cell lung cancer. Mol. Cancer. 9:198.
  144. 144.LiRWangHBekeleB. NYinZCarawayN. PKatzR. LStassS. AJiangF2006Identification of putative oncogenes in lung adenocarcinoma by a comprehensive functional genomic approach. Oncogene.1826282635
  145. 145.SchneiderCKingR. MPhilipsonLGenes specifically expressed at growth arrest of mammalian cells. Cell.198854787793
  146. 146.AmaldiFPierandrei-amaldiP1997TOP genes: a translationaly controlled class of genes including those coding for ribosomal proteins. In: Jeanteur P, editor. Progress in molecular and subcellular biology.18Springer-Verlag; Berlin, Germany:117
  147. 147.Mourtada-maarabouniMPickardM. RHedgeV. LFarzanehFWilliamsG. T2009GAS5, a non-protein-coding RNA, controls apoptosis and is downregulated in breast cancerGAS5 regulates apoptosis. Oncogene.28195208
  148. 148.GeeH. EBuffaF. MCampsCRamachandranALeekRTaylorMPatilMSheldonHBettsGHomerJWestCRagoussisJHarrisA. L2011The small‐nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis. Br. J. Cancer.10411681177
  149. 149.NakamuraYTakahashiNKakegawaEYoshidaKItoYKayanoHNiitsuNJinnaiIBesshoM2008The GAS5 (growth arrest-specific transcript 5) gene fuses to BCL6 as a result of t(1;3)(q25;q27) in a patient with B-cell lymphoma. Cancer Genetics and Cytogenetics.182144149
  150. 150.Askarian-amiriM. ECrawfordJFrenchJ. DSmartC. ESmithM. AClarkM. BRuKMercerT. RThompsonE. RLakhaniS. RVargasA. CCampbellI. GBrownM. ADingerM. EMattickJ. S2011SNORD-host RNA Zfas1 is a regulator of mammary development and a potential marker for breast cancer RNA.17878891
  151. 151.GuptaVKumarA2010Dyskeratosis congenita. Adv Exp Med Biol.685215219
  152. 152.RuggeroDGrisendiSPiazzaFRegoEMariFRaoP. HCordon-cardoCPandolfiP. P2003Dyskeratosis congenita and cancer in mice deficient in ribosomal RNA modification. Science.29925962
  153. 153.OnoMScottM. SYamadaKAvolioFBartonG. JLamondA. I2011Identification of human miRNA precursors that resemble box C/D snoRNAs. Nucleic Acids Res.3938793891
  154. 154.XiaoJ,LinH,LuoX,LuoX,WangZ. (2011).miR-605 joins p53 network to form a p53:miR-605:Mdm2 positive feedback loop in response to stress. EMBO J.30:5021.
  155. 155.SiomiM. CSatoKPezicDAravinA. A2011PIWI-interacting small RNAs: the vanguard of genome defence. Nat Rev Mol Cell Biol.12246258
  156. 156.IshizuHNagaoASiomiH2011Gatekeepers for Piwi-piRNA complexes to enter the nucleus. Curr Opin Genet Dev.21484490
  157. 157.Kazazian HH Jr2004Mobile elements: drivers of genome evolution. Science30316261632
  158. 158.SaitoKSiomiM. C2010Small RNA-mediated quiescence of transposable elements in animals. Dev. Cell.19687697
  159. 159.VaginV. VKlenovM. SKalmykovaA. IStolyarenkoA. DKotelnikovR. NGvozdevV. A2004The RNA interference proteins and vasa locus are involved in the silencing of retrotransposons in the female germline of Drosophila melanogaster. RNA Biol.15458
  160. 160.KalmykovaA. IKlenovM. SGvozdevV. A2005Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline. Nucleic Acids Res.3320522059
  161. 161.SavitskyMKwonDGeorgievPKalmykovaAGvozdevV2006Telomere elongation is under the control of the RNAi-based mechanism in the Drosophila germline. Genes Dev.20345354
  162. 162.LiCVaginV. VLeeSXuJMaSXiHSeitzHHorwichM. DSyrzyckaMHondaB. MKittlerE. LZappM. LKlattenhoffCSchulzNTheurkaufW. EWengZZamoreP. D2009Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies. Cell.137509521
  163. 163.AravinAGaidatzisDPfefferSLagos-quintanaMLandgrafPIovinoNMorrisPBrownsteinM. JKuramochi-miyagawaSNakanoTChienMRussoJ. JJuJSheridanRSanderCZavolanMTuschlT2006A novel class of small RNAs bind to MILI protein in mouse testes. Nature.442203207
  164. 164.GirardASachidanandamRHannonG. JCarmellM. A2006AGermline-specificclass of small RNAs binds mammalian Piwi proteins. Nature.442199202
  165. 165.BrenneckeJAravinA. AStarkADusMKellisMSachidanandamRHannonG. J2007Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell.12810891103
  166. 166.LauN. CSetoA. GKimJKuramochi-miyagawaSNakanoTBartelD. PKingstonR. E2006Characterization of the piRNA complex from rat testes. Science313363367
  167. 167.HouwingSBerezikovEKettingR. F2008Zili is required for germ cell differentiation and meiosis in zebrafish. EMBO J.2727022711
  168. 168.RobineNLauN. CBallaSJinZOkamuraKKuramochi-miyagawaSBlowerM. DLaiE. C2009A broadly conserved pathway generates 3’UTR-directed primary piRNAs. Curr. Biol.1920662076
  169. 169.AravinA. ASachidanandamRBourc’his D, Schaefer C, Pezic D, Toth KF, Bestor T, Hannon GJ. (2008A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. Mol. Cell.31785799
  170. 170.QiaoDZeemanA. MDengWLooijengaL. HLinH2002Molecular characterization of hiwi, a human member of the piwi gene family whose overexpression is correlated to seminomas. Oncogene.2139883999
  171. 171.TaubertHWürlPGreitherTKapplerMBacheMBartelFKehlenALautenschlägerCHarrisL. CKaushalDFüsselSMeyeABöhnkeASchmidtHHolzhausenH. JHauptmannS2007Stem cell-associated genes are extremely poor prognostic factors for soft-tissue sarcoma patients. Oncogene.2671707174
  172. 172.TaubertHGreitherTKaushalDWürlPBacheMBartelFKehlenALautenschlägerCHarrisLKraemerKMeyeAKapplerMSchmidtHHolzhausenH. JHauptmannS2007Expression of the stem cell self-renewal gene Hiwi and risk of tumour-related death in patients with soft-tissue sarcoma. Oncogene.2610981100
  173. 173.WangQ. EHanCMilumKWaniA. A2011Stem cell protein Piwil2 modulates chromatin modifications upon cisplatin treatment. Mutat Res.7085968
  174. 174.LuYLiCZhangKSunHTaoDLiuYZhangSMaY2010Identification of piRNAs in Hela cells by massive parallel sequencing. BMB Rep.4363541
  175. 175.CuiLLouYZhangXZhouHDengHSongHYuXXiaoBWangWGuoJ2011Detection of circulating tumor cells in peripheral blood from patients with gastric cancer using piRNAs as markers. Clin Biochem.4410501057
  176. 176.ChengJGuoJ. MXiaoB. XMiaoYJiangZZhouHLiQ. N2011piRNA, the new non-coding RNA, is aberrantly expressed in human cancer cells. Clin Chim Acta.41216211625
  177. 177.ChengJ,DengH,XiaoB,ZhouH,ZhouF,ShenZ,GuoJ. (2012).piR-823, a novel non-coding small RNA, demonstrates in vitro and in vivo tumor suppressive activity in human gastric cancer cells. Cancer Lett. 315:12-17
  178. 178.ReuterMChumaSTanakaTFranzTStarkAPillaiR. S2009Loss of the Mili-interacting Tudor domain-containing protein-1 activates transposons and alters the Mili-associated small RNA profile. Nature Struct. Mol. Biol.16639646
  179. 179.ShojiMTanakaTHosokawaMReuterMStarkAKatoYKondohGOkawaKChujoTSuzukiTHataKMartinS. LNoceTKuramochi-miyagawaSNakanoTSasakiHPillaiR. SNakatsujiNChumaS2009The TDRD9-MIWI2 complex is essential for piRNA-mediated retrotransposon silencing in the mouse male germline. Dev. Cell17775787
  180. 180.StruhlK2007Transcriptional noise and the fidelity of initiation by RNA polymerase II. Nature Struct. Mol. Biol.14103105
  181. 181.AmaralP. PMattickJ. S2008Noncoding RNA in development. Mamm. Genome.19454492
  182. 182.DingerM. EAmaralP. PMercerT. RPangK. CBruceS. JGardinerB. BAskarian-amiriM. ERuKSoldaGSimonsCSunkinS. MCroweM. LGrimmondS. MPerkinsA. CMattickJ. S2008Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation. Genome Res.1814331445
  183. 183.MercerT. RDingerM. ESunkinS. MMehlerM. FMattickJ. S2008Specific expression of long noncoding RNAs in the adult mouse brain. Proc. Natl Acad. Sci. USA.105716721
  184. 184.CawleySBekiranovSNgH. HKapranovPSekingerE. AKampaDPiccolboniASementchenkoVChengJWilliamsA. JWheelerRWongBDrenkowJYamanakaMPatelSBrubakerSTammanaHHeltGStruhlKGingerasT. R2004Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell.116499509
  185. 185.PonjavicJPontingC. PLunterG2007Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs. Genome Res.17556565
  186. 186.FaulknerG. JKimuraYDaubC. OWaniSPlessyCIrvineK. MSchroderKCloonanNSteptoeA. LLassmannTWakiKHornigNArakawaTTakahashiHKawaiJForrestA. RSuzukiHHayashizakiYHumeD. AOrlandoVGrimmondS. MCarninciP2009The regulated retrotransposon transcriptome of mammalian cells. Nat Genet.4156371
  187. 187.GuttmanMAmitIGarberMFrenchCLinM. FFeldserDHuarteMZukOCareyB. WCassadyJ. PCabiliM. NJaenischRMikkelsenT. SJacksTHacohenNBernsteinB. EKellisMRegevARinnJ. LLanderE. S2009Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature.458223227
  188. 188.PangK. CFrithM. CMattickJ. S2006Rapid evolution of noncoding RNAs: lack of conservation does not mean lack of function. Trends Genet.2215
  189. 189.ChengL. LCarmichaelG. G2010Decoding the function of nuclear long non-coding RNAs. Curr Opinion in Cell Biology.22357364
  190. 190.KugelJ. FGoodrichJ. A2012Non-coding RNAs: key regulators of mammalian transcription. Trends in Biochemical Sciences.37144151
  191. 191.KhalilA. MGuttmanMHuarteMGarber M Ray A. Rivea Morales D. Thomas K, Presser A. (2009Many human large intergenic noncoding RNAs associated with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci USA.1061166711672
  192. 192.ZhaoJSunB. KErwinJ. ASongJ. JLeeJ. T2008Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science322750756
  193. 193.RinnJ. LKerteszMWangJ. KSquazzoS. LXuXBrugmannS. AGoodnoughL. HHelmsJ. AFarnhamP. JSegalEChangH. Y2007Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell12913111323
  194. 194.TsaiM. CManorOWanYMosammaparastNWangJ. KLanFShiYSegalEChangH. Y2010Long noncoding RNA as modular scaffold of histone modification complexes. Sciences.329689693
  195. 195.MorrisK. VSantosoSTurnerA. MPastoriCHawkinsP. G2008Bidirectional transcription directs both transcriptional gene activation and suppression in human cells. PLoS Genet. 4:e1000258.
  196. 196.NaganoTMitchellJ. ASanzL. APaulerF. MFerguson-smithA. CFeilRFraserP2008The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science.32217171720
  197. 197.GuptaR. AShahNWangK. CKimJHorlingsH. MWongD. JTsaiM. CHungTArganiPRinnJ. LWangYBrzoskaPKongBLiRWestR. Bvan de Vijver MJ, Sukumar S, Chang HY. (2010Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature.46410711076
  198. 198.KogoRShimamuraTMimoriKKawaharaKImotoSSudoTTanakaFShibataKSuzukiAKomuneSMiyanoSMoriM2011Long non-coding RNA HOTAIR regulates Polycomb-dependent chromatin modification and is associated with poor prognosis in colorectal cancers. Cancer Res.7163206326
  199. 199.YapK. LLiSMunoz-cabelloA. MRaguzSZengLMujtabaSGilJWalshM. JZhouM. M2010Molecular interplay of the non coding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. Mol Cell.38662674
  200. 200.BeltranMPuigIPenaCGarcíaJ. MAlvarezA. BPenaRBonillaFDe HerrerosA. G2008A natural antisense transcript regulates Zeb2/Sip1 gene expression during Snail1-induced epithelial-mesenchymal transition. Genes Dev.22756769
  201. 201.WangXAraiSSongXReichartDDuKPascualGTempstPRosenfeldM. GGlassC. KKurokawaR2008Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription. Nature.454126130
  202. 202.FengJBiCClarkB. SMadyRShahPKohtzJ. D2006The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator. Genes Dev.2014701484
  203. 203.HuarteMGuttmanMFeldserDGarberMKoziolM. JKenzelmann-brozDKhalilA. MZukOAmitIRabaniMAttardiL. DRegevALanderE. SJacksTRinnJ. L2010A large intergenic noncoding RNA induced by53mediates global gene repression in the p53 response. Cell. 142:409-419.
  204. 204.BondC. SFoxA. H2009Paraspeckles: nuclear bodies built on long noncoding RNA. J Cell Biol.186637644

Written By

Gianpiero Di Leva and Michela Garofalo

Submitted: November 8th, 2011Reviewed: November 12th, 2012Published: January 24th, 2013