Non-coding RNAs: the riddle of the transcriptome and their perspectives in cancer

Introduction

The discovery of DNA structure, defined the central dogma of molecular biology with a widely acceptance in the scientific community. As far as central dogma of biology is concerned, the genetic information flows from DNA to RNA to protein. RNAs are molecules that have some similarity to DNA not only as far as structure is concerned but also in chemical composition. Transcription is the transfer of genetic code from DNA to RNA and takes place in the nucleus. RNAs then exit the nucleus into the cell body. mRNAs undergo translation in the cell body, which is the making of protein based on the code in the mRNAs. The last ones have the ability to carry the code for making proteins, so they also called “coding RNAs”. For many decades it was widely spread the idea that the majority of RNAs in our cells are mRNAs. However, recent studies have changed this “status”, suggesting instead that most RNAs do not code for proteins, and these non-coding RNAs (ncRNAs) might even hold the key to playing a great role or to furthering our understanding of human diseases. Understanding regulatory ncRNA is currently one of science’s most important challenges. Small non-coding RNAs (sncRNAs) retain a prospective role in addition to controlling the expression of most of our gene. Long non-coding RNA (lncRNA) represent the majority of transcription products, yet we know next to nothing of their significance. During the last decade, a tremendous number of ncRNAs, has been raised from anonymity in order to define as a category of genetic elements, leaving its sign on the field of tumor biology. Only a small rate of the human genome corresponds to protein-coding genes. The recent discovery, that most of our transcriptome is non-coding, is very promising. The comprehension of the biological role of this unknown RNA world undoubtedly represents the next great frontier in biology. Over the last 20 years, the intensified study of the human genome became the catalyst for a significant shift in our understanding about the way DNA operates. Complete sequencing of the human genome, within the framework of Human Genome Project (HGP), contributed to the identification and mapping of human genes. Moreover, the aforementioned international scientific consortium revealed that only a small portion, almost 2% of total DNA, is translated into proteins. As result, for a long time scientific community was holding fast to a “gene-centric” belief characterizing the majority (98%) of human DNA as “junk”. Reconsideration of the term “junk”, which was describing the non-coding part of the human genome, accomplished mainly through the results of Encyclopedia of DNA Elements project (ENCODE). This project provided with evidence supporting that most of the considered as “junk” DNA was pervasively transcribed and it could participate in the regulation of protein-coding genes by forming complex regulatory networks. Thus, the point of interest was relocated from genes to transcripts as the fundamental units of the genome (1).

ncRNAs are mainly divided into two categories based on their length, using as cutoff the 200 nucleotides (nt) length. ncRNAs <200 nt-long are referred as sncRNAs and include miRNA, siRNA, piRNA, snoRNA, snRNA and tRFs (2). ncRNAs longer than 200 nucleotides constitute an individual class of ncRNAs known as lncRNAs (Figure 1) (3). In general, ncRNAs consist the typical RNA form in mammalian cells, encompassing abundant and functional types such as rRNA and tRNA, various small RNA types such as microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), tRNA-derived fragments (tRF), small nucleolar RNA (snoRNA), small nuclear ribonucleic acid (snRNA) as well as lncRNA. However, the overrepresentation of ncRNAs in total RNA begs the question whether or not all these RNA molecules play a biological role or they constitute “junk RNA” (4). Simultaneously, the databases including annotated ncRNA transcripts [e.g., miRBase (5), lncRNAdb (6), NONCODE (7) etc.] are expanding constantly with novel sequences, mainly due to the technological advancement through the establishment of high-throughput next-generation (NGS) technologies (8,9). In particular, NGS has revealed many ncRNAs originating from protein-coding genes (10-16). Still, the transcription itself of several sequences and/or their identification in RNA-seq data does not establish a strong argument for them to be considered as biologically active ncRNA molecules. As result, the researchers need to implement strategies based on robust biochemical and/or evolutionary data to evaluate the putative functional role of novel ncRNA candidates (4).

Figure 1 A classification of RNA types showing the whole new world of RNAs playing important roles in diverse biological functions by modulating gene expression via antisense and miRNA-mediated controls.

With this review, we will attempt to provide a summarizing overview of the most important classes of lncRNA and sncRNA, as they emerged from the current literature. Moreover, we will discuss in brief about their biogenesis, their implication in cellular homeostasis and cancer development as well as their potential as cancer biomarkers and therapeutic targets.

miRNAs

miRNAs are short, single-stranded, approximately 22 nucleotides in length endogenous RNA molecules. Primary miRNA (pri-miRNA) transcripts are transcribed by RNA pol II and undergo two cleavage events until mature miRNAs occur. Firstly, Drosha, an RNAse III enzyme, with its cofactor the microprocessor complex subunit DGCR8, processes pri-miRNA transcripts to precursor miRNA hairpin transcripts (pre-miRNA) in the nucleus. Then, pre-miRNA translocated to the cytoplasm via Exportin-5 (XPO5), where another RNAse III enzyme called Dicer cleaves pre-miRNA to form mature miRNA molecules that assemble into RNA-induced silencing complex (RISC) inside P-bodies (Figure 2) (17).

Figure 2 Encoded in the genome as part of longer transcripts, pri-miRNA must undergo two processing events in order to generate an active “mature” miRNA. DROSHA cleave pri-miRNA in the nucleus to generate precursor miRNA (pre-miRNA). pre-miRNA are then exported to the cytoplasm via Exportin-5 where there are processed by DICER to generate miRNA.

Up to date, miRNAs are the most extensively studied sncRNA molecules because of their involvement in transcriptional and post transcriptional regulation of protein-coding genes. Specifically, miRNA 5’ seed region (between nucleotides 2–7) interact with regions within the 3'untranslated region (3' UTR) of messenger RNA (mRNA) leading to the degradation or repression of the targeted mRNA(s), depending whether or not a perfect miRNA/mRNA complementarity is achieved (18). Moreover, in silico predictions suggest that over 60% of protein-coding genes could be putative targets of miRNAs. By extension, it can be declared that the expression of protein-coding genes, at least for the vast majority of them, is somehow under the regulation of miRNAs (19). Therefore, essential cellular processes as cell proliferation, cell differentiation, cell migration, angiogenesis or apoptosis are monitored by a wide, complicated miRNA network (20). As a result, any malfunction relevant to the biogenesis pathway of miRNAs is strongly associated with malignant transformation and hence renders them as key players during tumor initiation, metastasis promotion and progression of the disease (21,22).

Over the past decade, findings from several studies revealed a broad suppression of miRNAs expression in tumors compared to healthy tissues indicating for a defective miRNA-biogenesis pathway in human malignancies (23,24). A plethora of mechanisms, including genomic amplifications, deletions within fragile chromosomal sites, mutations, and epigenetic regulation of miRNA expression are responsible for the extensive deregulation of miRNA expression during carcinogenesis (25). As result, due to the fundamental mechanism of action of miRNAs to regulate specific mRNAs, they function either as oncogenes or tumor-suppressors and depending on the cellular context and different mRNA targets (26,27).

Cancer is a multistep process characterized by the ability of tumor cells to sustain chronic proliferation and continuously expand, evading growth suppressors and apoptotic signals (28-30). Numerous miRNAs have been found deregulated during oncogenesis and have been implicated in proliferation of tumor cells (31). For example, miR-17/92 cluster, which was initially proposed as an oncomiR in diffuse large B cell lymphomas (32), has been presented to be frequently overexpressed and maintain oncogenic activity in a wide spectrum of carcinomas (33). Specifically, in lymphomas, a highly notable oncogenic collaboration has been illustrated between MYC and miR-17/92. In particular, MYC is a transcriptional regulator that activates miR-17/92 cluster expression. As a result of miR-17/92 overexpression, expression of chromatin regulatory genes (SIN3B, HBP1, and BTG1) and the proapoptotic BCL2L11 gene is suppressed, contributing to survival maintenance and self-renewal of tumor cells (34). Furthermore, E2F transcription factor 1 (E2F1) is negatively regulated by miR-17/92, provoking attenuated E2F-induced apoptosis and simultaneously contributing to proliferative signal by promoting E2F transcription factor 3 (E2F3) expression (35). Members of the miR-17/92 cluster and its paralogue miR-106a/363, such as miR-92a-3p and miR-20b-5p, along with miR-155-5p have pivotal roles in other B-cell malignancies, including chronic lymphocytic leukemia (36-38). In particular, miR-155-5p regulates important transcription factors, such as E2F2 and hypoxia-inducible factor 1 (HIF1) in leukemic cells (39,40).

Hallmarks of carcinogenesis include invasion and metastasis of malignant cells as well as intense tumor angiogenesis as responding to the enormous needs for oxygen and nutrients (41). The miR-200 family members have been proposed as crucial regulators of multiple genes that are responsible for maintaining the epithelial polarity, like Zinc Finger E-Box Binding Homeobox 1 (ZEB1) and Zinc Finger E-Box Binding Homeobox 2 (ZEB2), which are controlling cadherin 1 (CDH1; also known as E-cadherin, ECAD) expression, and they actively participate in epithelial to mesenchymal transition (EMT) (42,43). Concerning the facilitation of angiogenesis by miRNAs, we could note the case of miR-126 which directly targets regulators of RAS/RAF1/MAPK pathway, crucial in angiogenic signaling [e.g., sprouty-related EVH1 domain-containing 1 (SPRED1)] (44).

Nowadays, the study of miRNA expression is mainly performed via the “gold standard” quantitative polymerase chain reaction (qPCR) followed by hybridization methodologies such as microarrays, while recent advancements in the era of NGS enabled high-throughput RNA-seq as an alternative for the in-depth analysis of miRNAs (45). As the knowledge regarding miRNAs is expanding more evidence depict the great diagnostic, prognostic and predictive potential of this novel class of biomarkers advocating for their active involvement in more and more daily clinical applications. Altered expression levels of miRNAs between malignant tumors and healthy tissues facilitate cancer diagnosis, ameliorate tumor staging and inform clinicians about relapse risk and/or disease progression as well as therapeutic efficacy, reducing the unwanted under- or overtreatment. The prognostic and predictive value improves by the synergy of more than two miRNAs, encouraging the development of multipurpose miRNA signatures as diagnostic or prognostic tools (46-48). For instance, combination of miR-15a-5p, miR-16, miR-24-3p, miR-28-5p, miR-34a, miR-96, miR-182, and miR-224, which have been proposed as molecular biomarkers with significant prognostic value in colorectal cancer (CRC); has already been proposed as a prognostic signature in this malignancy (49-57); another very important miRNA that could be added in this molecular signature is miR-21, a predictor of metastatic tumor potential in this cancer (58). Similar signatures consisting of miRNAs have been proposed in prostate cancer (59,60), laryngeal squamous cell carcinoma (61), and other human malignancies (62). Multiparametric panels of biomarkers could also integrate protein-coding transcripts produced by cancer-related genes such as the apoptosis-related genes BCL2L12 (63-67), BCL2 (68,69) and BAX (70,71), DDC (72-75), transcription factors such as HIF1 (39), and/or other mRNAs with significant prognostic value in human malignancies (76-83). Besides that, miRNAs can stably circulate in human body fluids (e.g., plasma, urine etc.), permitting their easy quantification paving the way for the development of non-invasive assays in the era of personalized medicine (84,85). Moreover, proteins that are targeted by important miRNAs could also participate in such multiparametric panels of biomarkers (86-89), along with clinical markers (90-92).

tRFs

tRFs constitute novel sncRNA molecules generated by specific cleavage of tRNA transcripts. There are two classes of tRF based on their length and their position of origin in primary or mature tRNA. The first includes stress induced tRFs, known as tRNA halves (tiRNAs or tiRs), generated by a specific cleavage by angiogenin within the anticodon loop of mature tRNAs to produce fragments ranging from 31 to 40 nucleotides. There are two subclasses of tiRNAs depending whether they include the 5’ or 3’ part of anticodon loop after cleavage (93). Interestingly, these angiogenin-produced fragments differ from other sncRNAs, including miRNAs and other tRF classes, by carrying a 5’hydroxyl group. tiRNA production by stressed cells repress translation and modulate intrinsic stress-response program of the cells. Moreover, they interact with Argonaute (AGO) protein members (e.g., AGO2) to form complexes participating in RNA interference (RNAi) silencing pathway (94). Recently, overexpression of tiRNAs in a sex hormone-dependent manner has been identified in breast cancer and prostate cancer cells positive for estrogen and androgen receptors (ARs) respectively. These sex-hormone dependent tiRNAs enhance cell proliferation, however, the mechanism of their action needs to be further investigated (95).

The second tRF class includes smaller tRNA fragments 14–30 nucleotides long, from the ends of mature or primary tRNAs which are under the spotlight of scientific community due to their size and similarity to miRNAs. Based on their mapping to 5’ or 3’ ends of tRNAs or 3’ ends of primary tRNAs are divided in three subtypes tRF-5, tRF-3 and tRF-1 respectively (96). The molecular mechanism and the enzymes that participate to the cleavage of tRNAs producing tRF-5 and tRF-3 are still unknown. At the same time, a Drosha or Dicer dependent tRNA-process mechanism has been excluded due to the maintenance of tRF abundance in experiments using Dicer and Drosha knock-out cells. Regarding their cellular distribution tRF-5 can be found in the nucleus whereas tRF-3 are predominantly gathered in the cytoplasm (97). These data are further supported by studies indicating a strong association of tRF-5 with Piwi proteins in monocytes to repress CD1A expression epigenetically through the methylation of its promoter (98). Moreover, it has been proved that tRF-3s interact with AGO proteins and act in a miRNA-like way to regulate crucial oncogenes or tumor suppressors (99).

On the contrary, tRF-1 consists a more variable tRF subclass generated by a 3’ cut of tRNA precursors from RNase Z (100). Also, tRF-1 interact with members of Piwi and AGO protein families and potentially regulate gene expression in a piRNA- or miRNA-like manner (100,101). Representatives of tRF-1 class have been found to accelerate proliferation in prostate cancer cell lines (96), or more recently to associate with the development of an aggressive CLL phenotype by regulating T cell leukemia/lymphoma 1A (TCL1A) gene (102).

Recently, tRF-2 (103) and internal tRFs (i-tRFs) (104) have been proposed as novel classes to describe other abundant tRFs. tRF-2 derived from anticodon stem loop of tRNAs and bind to Y-box binding protein 1 (YBX1) in breast cancer cells. YBX1 is a critical RNA-binding protein for the stabilization of multiple oncogenic transcripts (103). Additionally, in silico analysis of available (TCGA) datasets revealed a distinct category of tRFs, referred as i-tRFs, that spans entirely internal to mature tRNA sequences, exhibiting a cell type dependent expression and discriminating breast cancer histological subtypes (104).

piRNAs

P-element-induced wimpy testis (Piwi)-interacting RNA (piRNA) are 21–36 nucleotides single stranded RNA molecules and represent the largest group of sncRNAs. They are abundant in spermatogenic cells and they have been found to be critically involved in germline development and functions (105). In contrast to miRNA or siRNA mode of action, targeting transcripts directly, piRNAs interact with Piwi proteins, a subfamily of AGO proteins, to mediate epigenetic silencing (106). However, recent studies using Drosophila and mice as model organisms propose a potential miRNA-like function for piRNAs in the cytoplasm (107,108). piRNAs are mainly responsible for preserving genome integrity through the silencing of transposable elements (TE) (109).

Biogenesis of piRNAs has not been fully understood yet, thus several mechanisms have been proposed. The primary maturation mechanism involves the cleavage by Piwi proteins of a long primary RNA, transcribed from genomic regions identified as piRNA clusters (109). Ping-pong amplification cycle is another well-studied mechanism, characteristic of piRNAs, which bridges the gap between piRNA biogenesis and target silencing. In Drosophila germ cells a primary piRNA is associated with aubergine protein (AUB) to detect and slice active TEs. The aforementioned cleavage produces the 5' ends of new piRNA in sense with the cleaved TEs. Next, they are loaded into AGO3 and further maturate by trimming of their 3' ends. As result, new antisense piRNAs, similar to the original, are produced and target TEs (110).

piRNAs are functional RNA molecules implicated in gene silencing, thus raising the interest of scientist to define their potential role in human malignancies. Expression of oncogenes or tumor-suppressors carrying transposon-derived sequences in their 3’UTR could be modulated by piRNA (107). Deregulated piRNA fail to control and suppress the activity of TEs increasing genomic instability and promoting mutagenesis, leading to the development of aggressive cancer phenotypes (111). Moreover, Martinez et al. (112) recently revealed tissue specific piRNA expression patterns and specific piRNA-signatures between cancerous and healthy samples using The Cancer Genome Atlas (TCGA) datasets.

In breast cancer (BC) piR-36011, piR-31106 and piR-36717 have been found to differentially expressed in hormone-responsive BC, but also between cancer and normal breast tissue specimens (113). Additionally, four piRNA (piR-4987, piR-20365, piR-20485 and piR-20582) were confirmed to be upregulated in breast cancer using deep sequencing data and qPCR. Notably, piR-4987 was associated with positive lymph node status, indicating the great potential of piRNA as biomarkers in BC (114). A very recent study illustrated piR-1245 as a frequently over-expressed piRNA in CRC. Furthermore, patients with elevated piR-1245 levels were susceptible to metastasis and demonstrated lower overall survival (OS) (115). piR-651 has been proposed as a putative oncogenic piRNA as it has been found highly upregulated in gastric cancer (GC) tissues with this upregulation to be also validated in different human cancer cell lines including hepatic, cervical, breast, mesothelioma and lung (116). Similarly, piR-823 found to be upregulated in multiple myeloma (MM) patients and cell lines. Moreover, it correlated positively with the stage of the disease and characterized as a crucial molecule in the process of DNA methylation and an important regulator of myelomagenesis by stimulating bone marrow neo-angiogenesis (117). Interestingly, piR-651 and piR-823 have been used as peripheral blood biomarkers for the detection of circulating cancer cells of GC patients, able to discern patients with GC from the healthy individuals (118).

snoRNAs

snoRNAs are a distinct regulatory class of sncRNA (60–250 nucleotides) operating as guide molecules during post-transcriptional chemical modifications, such as 2’-O-methylation and pseudouridylation, of rRNA or other RNA molecules. Distinct sequence motifs and secondary structure classify snoRNA as C/D box, H/ACA box or small Cajal body-specific RNAs (scaRNA) (119). Box C/D snoRNAs (60–200 nucleotides) are distinguished by the presence of two highly conserved canonical motifs referred as C box (RUGAUGA motif, where R is a purine) and D box (CUGA motif). These snoRNAs catalyzing the site-specific 2’-O-ribose methylation of rRNA residues. Box H/ACA snoRNAs are longer than box C/D snoRNAs, ranging from 120 to 250 and guiding pseudouridylation of rRNA residues. A two-hairpin structure connected by an H box region (ANANNA, N corresponds to nucleotide) is characteristic for box H/ACA snoRNAs. Moreover, ACA trinucleotide is located three nucleotides upstream of the 3’-end of the snoRNA (120). ScaRNA are larger than the other snoRNA classes, accumulated in Cajal bodies and they characterized by the presence of both C/D and H/ACA boxes along with a CAB box (UGAG motif) that functions as a Cajal-body specific localization signal (121).

Besides their role in modification and maturation of rRNA, strong evidence has emerged concerning their potential role in human malignancies. The significance of these data is enhanced by several studies indicating a potential miRNA-like function for some small RNAs, derived from snoRNAs, after Dicer processing and interacting with AGO proteins (122,123). snoRNA U50 has been identified as a candidate tumor suppressor, downregulated in prostate and breast cancer (124,125).

Liao et al. (126) presented six deregulated snoRNAs (SNORA42, SNORD33, SNORD66, SNORD76, SNORD78, and SNORD73B) in plasma of non-small-cell lung cancer (NSCLC) patients. Of them SNORA42 was proposed to be implicated in tumorigenesis through two different pathways, a p53-dependent and a p53-independent. Notably, NSCLC patients with elevated SNORA42 expression succumbed earlier from the disease (127). In the same context, twenty-two snoRNAs were identified to demonstrate alterations specific to cancer stem cells of NSCLC patients (128). Human cell line screening of the most representative leukemic groups, including acute myeloid leukemia (AML), pre-B-acute lymphoblastic leukemia (ALL) and T-acute lymphoblastic leukemia (ALL) revealed distinct snoRNA expression patterns between various leukemic groups, however the discriminatory potential of these snoRNA, as well as their involvement in pathobiology of human leukemias, need to be further elucidated (129).

lncRNAs

Approximately 16,000 genes of the entire gene code databases belong to an important class of ncRNAs greater than 200 nucleotides in length and this number is increasing with a great rate. Very few of these RNA molecules have been characterized at all apart from their detection in different sample types. What we do know, is that expression of many of these lncRNAs is highly tissue specific and many are detected only under certain stress conditions. These fascinating molecules called lncRNAs. The last ones can be exonic, intergenic, in enhancer regions, or in regions distal to protein-coding genes. Similar to mRNAs, lncRNAs are transcribed by RNA polymerase II (RNApol II), carry single nucleotide polymorphisms (SNPs), can undergo alternative splicing, may have 5’ caps, and are usually polyadenylated. It is mentionable that under estimations the majority of lncRNAs has more than two exons, and can have secondary and tertiary structures (130). lncRNAs can function in multiple ways. They can act as scaffolds (for example NEAT1 and HOTAIR have the ability to act in trans) or guides (for example Xist, Kcnq1ot1, Airn have the ability to act in cis whereas HOTAIR acts in trans). But in addition to these two simple ostensible mechanisms, there is a plenty number of others by which lncRNAs can function and influence on cell operation. Finally, they present the ability to act as enhancers (e.g., eRNAs, in cis), reservoirs (e.g., H19) but also as decoys (e.g., Tsix, MALAT1). A tremendous increasing number of experimental studies, are providing evidence that lncRNAs mediate human disease pathogenesis, thereby challenging the concept, that protein-coding genes, are the sole contributors to the development of human disease pathogenesis. As the scientists investigate, the encoding RNA is carrying out varied cell operations in both cytoplasm and the nucleus very often involved in regulation of gene expression.

A plenty number of lncRNAs are located exclusively in the cytoplasm and others in nuclear. The last ones appeared to be heavily involved in genetic regulation of gene transcription. They recruit and guide proteins involved in modifying chromatin structure. Many lncRNAs are nuclear retained or have long residence time in the nucleus and as a consequence are often inefficient targets of RNAi. In addition, scientific studies have been descripted that a plenty number of lncRNAs are often expressed from complex loci with not only overlapping sense but also and antisense transcription (131).

lncRNAs & human malignancies

It is being recognized that certain single-nucleotide polymorphisms (SNPs) are associated with tumor risk. Large-scale data analysis from cancer genome-wide association studies indicates that the majority of SNPs associate with non-coding genes (132,133). The majority of recurrent mutations in somatic cells, copy number alterations, and tumor-related SNPs are related to ncRNAs (134-136), and the presence of risk SNPs may modulate the expression of corresponding ncRNAs. Among this number of non-coding genes, lncRNAs are emerging as a new team of indispensable members involved in the development and progression of tumor (137-140).

Moreover, the dysregulation of a number of lncRNA targets, has correlated with the prognosis and diagnosis of a plenty number of cancer types including prostate cancer (141,142), lung cancer (143), and breast cancer (144-146), among other tumor types (147,148), as well as being linked to detention against chemotherapy and targeted therapy (149-152). Correlation with a great number of analyses, indicates that these molecules are upregulated in cancer cells that are resistant to DNA damage inducers (153-156), targeted therapies or anti-hormone therapies (141,157-159). Loss-of-function studies using small hairpin RNA-based knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)/cas9-mediated genetic depletion indicate that lncRNAs facilitate cancer cell growth, cell mobility and apoptosis detention, (150,160,161). The expression of HOTAIR activates estrogen receptor (ER) target transcription program and contributes to resistance to tamoxifen (161). Gain-of-function studies suggest that increased expression of lncRNAs enhances cell viability during drug treatment (153,158,162). lncRNA derives from several sources including the antisense strand of the protein coding sequence, intronic transcription, intergenic regions, or alternative splicing (163,164). A considerable percentage of known lncRNAs either reside within the cytosol or shuttle between the nucleus and the cytoplasm (165).

Recent studies present that these types of cytoplasmic lncRNAs play a great functional role in modulating mRNA translation and decay in a base-pairing dependent manner (166-168) or by competing with miRNA-mediated or a protein- mRNA decoy (169). In addition, cytoplasmic lncRNAs have been shown to regulate cytoplasmic protein trafficking from the cytosol to the nuclear areas for transcriptional activation (170). Current studies have also indicated that lncRNAs may associate with proteins, metabolic intermediates and cellular lipids. Although still largely unexplored, it has been suggested from scientists that lncRNAs are part of an essential intracellular signaling pathway. Novel type optical aspects into the regulatory roles of lncRNAs in tumor for governing new type mechanisms and pathways by which tumor cells acquire their metastatic and invasiveness properties serve as the basis of a new insight in the battle against tumorigenesis. This empathy of lncRNAs in tumor signaling should stimulate new directions for future research therapeutic options that focus on lncRNAs as novel tumor diagnostic, prognostic markers and therapeutic targets.

lncRNAs in prognosis & cancer therapy

A mentionable number of lncRNAs is deregulated in cancer and contribute to oncogenesis. In a plenty number of tumors, several lncRNAs as well as ncRNAs being transcribed from protein-coding genes (nonsense-mediated mRNA decay candidates) have been reported to be overexpressed and proposed as biomarkers (171-173). For example, several lncRNAs (GAS5 or H19) have been reported to be frequently or consistently overexpressed in urothelial carcinoma and have been proposed as individual diagnostic or prognostic biomarkers; some were moreover demonstrated to influence survival, proliferation, migration and other cancer-relevant properties of UC cell lines (174).

From a clinical aspect, lncRNAs serve as novel promising therapeutic targets. A plenty number of therapeutic strategies have been developed to target and to manipulate lncRNAs. Antisense oligonucleotide (ASO)-based strategies that downregulate the transcripts of lncRNAs via RNaseH-dependent degradation are under active investigation (175). Alternatively, nanoparticle-delivered siRNAs have been developed to knockdown lncRNAs in vivo via Argonaute- and Dicer-dependent RNA silencing (176-178), which have been evaluated in many types of models and have been found to inhibit tumor genesis and distant metastasis (178,179). Small-molecule inhibitors to block lncRNA-protein interactions or interfere with lncRNA-protein complex formation, are also on the rise. Interestingly, many types of tumors frequently become resistant to administered chemotherapeutic agents. In these chemotherapy-resistant cancers, dysregulated lncRNAs have the ability to contribute significantly to the development of this resistance (175). During clinical trials, combinations of pathway-specific inhibitors integrated with an lncRNA-directed strategy could provide maximum efficacy in treating human tumors, which is under active investigation.

Targeting lncRNAs using a variety of technologies, including ASO-based strategies, siRNAs and small molecular inhibitors should be evaluated for their effects on tumor initiation, progression or metastasis and response to therapy ASOs, including duplex RNA (180), ASO gapmers (179), and locked nucleic acids (LNAs) (181) present the ability of bind base pairing with lncRNA transcripts. The RNA-DNA duplex triggers RNase-H-dependent cleavage (182). The modern modified generation of ASOs incorporates chemical/biological modification of the backbone, constructed by sugar to improve in vivo stability and in binding affinity (183). S-constrained ethyl (cEt) modifications (184) and LNAs (181,185,186) have been advanced to pre-clinical experiments (187-189). The major characteristic of LNAs which are constructed nucleotides is the fact that contain an extra covalent bond between the 4’-C and 2’-O of the ribofuranose ring (190,191). The LNA-DNA-LNA gapmers have the ability to pair with RNA targets, which can be used to silence RNA targets not only in animal models but also in cell-line-based experiments. A similar purpose as LNAs can serve by the incorporation of bridged nucleic acid (BNA) monomers (192). In order to study the effects on cancer growth and metastasis the immediate application of ASOs to knockdown lncRNAs in vivo has been tested in a plenty of tumor models (193). LNAs present the great ability to target plasmacytoma variant translocation 1 (PVT1) and this fact has as a result the sensitization of cervical cancer cells to cisplatin, substantiating the effectiveness of combinatorial treatment (194). Clinical trials or scientific experiments using LNAs targeting AR (195) or oncoprotein Bcl-2 (196), have presented a promising result and this has as a result to be under consideration. Beyond its multiple uses in tumor, LNAs have also been proposed to improve the status of patients with cardiovascular disorders (197), neuronal diseases (198), kidney disorders (199), and other human diseases. Studies have indicated that, dioleoyl phosphatidylcholine (DOPC)-based nanoliposomes have been developed and constructed in order to deliver the nucleotide based-therapeutics (miRNA, siRNA, ASOs, and lncRNA) for clinical trial (196,200-207). Experiments have presented that a single injection of DOPC-nanoliposomal siRNAs can promote or influence the expression of target proteins for four days in mice tumors (200,201). This unique administration promotes a significant repression in the levels of expression of the gene targets (for example, BCL2, KRAS, eEF2K, miR34a, miRs155, and JAK2) and in size as far as is concerned the tumors in rodent models and preclinical models of human tumors, including many kinds of tumor models (e.g., xenografts or orthotopic models) (196,200,201,203-205,208,209).

Last but not least, RNA molecules consist targets for small-molecule inhibitors. Via high-throughput screening, scientists can identify small-molecule compounds that may potentially inhibit RNAs (210-212). There are a great number of serious efforts in order to establish platforms and methods to aid the design and identification of small molecule inhibitors for oncogenic ncRNAs but with non-mentionable results (213), fact that will facilitate the huge development of clinical or pharmaceutical agents that target lncRNAs molecules.

Conclusions

Future studies on the regulatory and biological roles of sncRNAs and lncRNAs in cancer signaling will define the future of the field. Although a huge list of ncRNAs has been identified thus far, it has been a strenuous task to demonstrate the functional relevance of ncRNAs in cancer. To answer this problem, thorough examinations of ncRNAs candidates involved in cancer signaling pathway need to be conducted to reveal the physiological relevance of ncRNAs in cell apoptosis, survival, metastasis, and metabolism. Cellular and xenograft models have been the common means of studying the roles that ncRNAs play in cancer and are useful tools in cursory evaluations of their functions. However, conclusions that are more definitive will require representative in vivo models of cancer, such as genetic models that better recapitulate the tumor microenvironment. It will be crucial to determine if tissue-specific expression of ncRNAs can induce tumor formation, which can then be blocked by targeting the ncRNAs. Identification of the specific ncRNAs that function in various human cancer types has enabled the development of ncRNA-based clinical applications such as biomarkers for diagnosis, prognostic indicators, drug sensitizers, and therapeutic targets. The ncRNAs profile of each human cancer type should be systematically investigated to improve clinical outcomes for cancer patients by engendering a personalized approach to medicine.

Acknowledgements

The research presented was carried out within the framework of a Stavros Niarchos Foundation grant to the National and Kapodistrian University of Athens.

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

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