Cancer is one of the most serious diseases around the world and it is the third leading cause of death, exceeded only by heart and infectious diseases . There are five major steps for cancer development: initiation, promotion, malignant conversion, progression, and metastasis . Cancer is result of process, where somatic cells mutate and escape the controlled balance of gene expression and cellular networks that maintain cellular homeostasis, which normally prevent unwanted expansion. Perturbations in these pathways results in cellular transformation, where cancer cells differ from their normal counterparts in many characteristics, as is loss of differentiation, increased invasiveness, and decreased drug sensitivity [3-4]. There are six primary hallmarks of cancer: unlimited cell proliferation, autonomous growth without the need of external signals, resistance to growth inhibitory signals, escape from apoptosis the ability to recruit new vasculature and increased tissue invasion and metastasis . The formation of cancer is therefore fundamentally genetic and epigenetic disease requiring accumulation of genomic alterations to inactivate tumour suppressor and activate proto-oncogenes . These results in combined interaction of both tumour suppressors, that are not able to inhibit tumour development and protect cells against mutation that initiate transformation, and cancer inducers, which promotes cancer development as initiators of cellular transformation. When cells exhibit abnormal growth and loss of apoptosis, it usually results in cancer formation .
Genetic studies have revealed the mutational and epigenetic alterations of protein-coding genes that control DNA damage response, growth arrest, cell survival and apoptotic pathways . Until recent years ago, the central dogma of molecular biology was that genetic information is stored in protein-coding genes with RNA as an intermediate between DNA sequence and its encoded protein . Recent studies suggest that advanced stages of cancer are possessing more severe molecular perturbations and that this could be due to function of non-coding RNAs (ncRNAs), which were previously known only to have infrastructural functions (as ribosomal RNA, transfer RNA, small nuclear and nucleolar RNA). Eukaryotic genomes are extensively transcribed into thousands of long and short ncRNAs, which are group of endogenous RNAs that also function as regulators of gene expression. They are involved in developmental, physiological as well as pathological processes [7,8].
However, in this review, the following characteristics of ncRNA in human cancers will be summarized: (i) the current understanding of the critical role that lncRNAs and miRNAs may play in cancer as tumour suppressors; (ii) outline current knowledge about some specific lncRNA and miRNAs and their target genes in cancer; (iii) highlight their potential as biomarkers for patho-histological subtype classification; and (iv) highlight their potential as biomarkers and as circulating biomarkers and therapeutic targets in cancer.
Since the majority of research regarding regulatory ncRNAs as tumour suppressor was performed on miRNAs and in lesser extend on lncRNAs/lincRNAs, will this review further focused on these two groups of ncRNAs.
2. Brief overview of non-coding RNAs (ncRNAs)
Of transcribed eukaryotic genomes, only 1-2 % encode for proteins, whereas the vast majority are ncRNAs that are in more or less functional transcripts. The regulatory ncRNAs are important regulators of gene expression in many eukaryotes and are involved in a wide range of functions in eukaryotic biology [8,9].
Based on their function, ncRNAs can be divided into two groups. First is infrastructural group, with ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). Second is regulatory group, with microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), small-interfering RNAs (siRNAs), long non-coding RNAs (lncRNAs), large intergenic non-coding RNAs (lincRNAs), promoter-associated small RNAs (PARs), repeat-associated short interfering RNAs (rasiRNAs) and enhancer RNAs (eRNAs) [8,9]. Recent findings suggest that some structural ncRNAs (e.g. snoRNAs) not only have infrastructural function but have regulatory as well .
Based on length, the regulatory ncRNAs can be divided in two groups: larger than 200 nucleotides (nt) are lncRNA, lincRNA, eRNA, whereas the others are smaller than 200 nt, with exception of PARs that are 16-30 nt long or up to 200 nt. Distinct classes of small RNAs are distinguished by their origins, and these are: snRNAs, snoRNAs, miRNAs, piRNAs, siRNAs, and rasiRNAs [8,9].
miRNAs and snoRNAs share similarities in processing pathways and protein interaction partners, genomic organization and location, as well as levels of conservation. However, similarities in sub-cellular localization have been also observed, since large proportion of human mature miRNAs have been detected in the nucleus as well as a subset of small RNAs derived from snoRNAs have been detected in the cytoplasm .
The most widely studied and characterized of all the regulatory ncRNAs are miRNAs. The roles of regulatory ncRNAs, other than miRNAs, in the mediating transcriptional regulation, chromatin remodelling, post-transcriptional regulation, and other processes are less well understood. The contexts of gene regulation by ncRNAs in non-human systems provided insights into how these processes could function in human cells. Regulatory ncRNAs are involved in diverse cellular pathways, such as development and stem cell maintenance, response to stress and environmental stimuli, regulating chromatin structure and remodelling, chromosome architecture and genome integrity, transcription (positive or negative impact), and post-transcription processing (splicing, transport) and most commonly mRNA stability (translation, degradation) [8,9]. Some ncRNAs trigger different types of gene silencing that are collectively referred to as RNA silencing or RNA interference .
RNAi is RNA-guided regulation of gene expression, historically known by other names, including post-transcriptional gene silencing. It is believed to be an evolutionary conserved mechanism in response to presence of foreign dsRNA in the cell. A key step in this silencing pathway is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. The enzyme Dicer, which initiates the RNAi pathway, cleaves dsRNA to short double-stranded fragments of 20–25 base pairs (bp), named siRNAs. siRNAs usually possess perfect complementarity to the mRNA of target gene, thus causing its degradation. When the dsRNA is exogenous, coming from infection by a virus with RNA genome or laboratory manipulations, the RNA is imported directly into the cytoplasm where it is cleave by the Dicer. On other hand, the initiating dsRNA could be result of endogenously expressed RNA-coding genes from the genome. Some of small regulatory RNAs are processed in a similar way or with components of RNAi pathway .
2.1. Brief introduction to miRNAs
miRNAs are endogenously expressed small (~22 nt), single-stranded ncRNAs. It is predicted that they constitute ~1-5 % of human genes [1,12] and in an update from August 2012, miRBase v19 was released with a list of 2019 unique mature human miRNAs. miRNAs are encoded as a single gene or gene clusters, with some of miRNA clusters being co-regulated and co-transcribed. Intergenic miRNAs are transcribed as an independent transcription unit, as a monocistronic, bicistronic or polycistronic primary transcripts . Up to 60 % of currently known miRNAs are proposed to be from intronic sequences of either protein coding or non-coding transcription units and suggestion has been made that some miRNAs are also encoded in antisense DNA, which is not transcribed to the mRNA. Intronic miRNA are preferentially transcribed in the same orientation as the host gene and are together with their host transcripts co-regulated and co-transcribed from the same promoter. They are processed from introns, as are many snoRNA. Within the genome, there might be more than one copy of particular miRNA [13,14].
miRNAs expression is determined by intrinsic cellular factors and diverse environmental variables . As for protein-coding genes it is known, that regulation of miRNA transcription and expression depends on transcription factors and epigenetic mechanisms (e.g. p53, Myc, and myogenin). In general, from genes encoding miRNAs is transcription guided by RNA-polymerase II (Pol II). Resulting primary transcript (several hundred bases to several kilobases), named
Numerous alternative pathways differing from canonical miRNA biogenesis pathway have been described recently and subset of several diverse longer non-coding RNAs can serve as precursors for miRNAs [10,16]. As an example, intronic miRNAs presumably bypass Drosha cleavage, since through
The functional role of miRNA varies, but the primary mechanism of miRNA action in mammals is believed to be base-pairing to 3'-UTR of target mRNA followed by inhibition of mRNA translation (when base pairing between these two molecules is incomplete) or deadenylation and degradation (perfect complementarity of miRNA:mRNA binding) . Especially in animals, the primary mechanism of miRNA action is reducing mRNA translation and each miRNA can inhibit the translation of as many as 200 target genes. In addition, mRNA can be regulated by more than one miRNA. The cooperative action of multiple identical (multiplicity) or different miRNPs (cooperativity) appears to provide the most efficient translational inhibition. Additional mechanism to increase the specificity of miRNAs is combinatorial control of gene expression, which may be also provided by a set of co-ordinately expressed miRNAs. Proteins or mRNA secondary structures could restrict miRNP accessibility to the UTRs, or may facilitate recognition of the authentic mRNA targets [12,13,18,19].
There is the prospect that some miRNA might specify more than just post-transcriptional repression [9,13]. miRNAs may also target promoter to regulate transcription through epigenetic mechanism. miRNAs have been paradoxically also shown to up-regulate gene expression by enhancing translation under specific conditions .
Translational repression, as major mechanism of miRNAs, may in normal cell conditions occur in different ways: as switch off the targets, that is for mRNAs that should not be expressed in a particular cell type, the protein production is reduced to inconsequential levels; as fine-tuners of target expression, that is when miRNAs can adjust protein output for customized expression in different cell types; as neutralizers of target expression, that is when miRNAs act as bystanders, where down-regulation by miRNAs is tolerated or reversed by feedback processes . Role of miRNA can be further divided in three paradigms: combinatorial control (defined as cooperativity), cell-to-cell variation, specific (tissue-specific and/or cell-type specific) and housekeeping functions .
Despite the large number of identified miRNAs, the scope of their roles in regulating cellular gene expression is not fully understood . It is believed that miRNAs through negative gene regulation influence at least 50 % of genes within the human genome . Expression profiling of many miRNAs in various normal and diseased tissues have demonstrated unique spatial and temporal expression patterns. Many miRNAs are important at distinct stages of development and have been found to regulate a variety of physiological and pathological processes . miRNAs are involved in a numerous biological processes, such as stem cell division and developmental timing, proper organ formation, embryonic pattering and body growth, proliferation and differentiation, apoptosis, epithelial-mesenchymal-transition (EMT), cholesterol metabolism and regulation of insulin secretion, resistance to viral infection and oxidative stress, immune response etc. [2,11]. All these effects may occur by regulating or being regulated by the expression of signalling molecules, such as cytokines, growth factors, transcription factors, pro-apoptotic and anti-apoptotic genes . With all different genes and expression patterns, it is reasonable to propose that every cell type at each developmental stage might have a distinct miRNA expression profile.
Up to date, over 2000 human miRNAs have been identified and this number is still growing. All annotated miRNAs are collected in miRBase . The first step in miRNA target identification is usually defining reciprocally regulated miRNA-mRNA or miRNA-protein. Since miRNAs target mRNA mainly by incomplete base-pairing, many computational methods have been recently developed for further identifying potential miRNA targets . Most of these methods search for three criteria in predicting miRNA target genes: first, multiple conserved regions of miRNA complementarities within 3'-UTR of target mRNA (evolutionary conservation); second, interaction between seven consecutive nucleotides in the target mRNAs 3'-UTR and the 1-8 nt (“seed sequence”) at the 5' miRNA end; third, stability of base pairing and predicted binding energy. Further complicating target site prediction in mammals is the fact that not all 3'-UTR sites with perfect complementarities to the miRNA seed nucleotides are functional. Moreover, mRNAs sites with imperfect seed complementarities can themselves be very good miRNA targets [24,25]. Bioinformatics is therefore much noisier and more prone to false positive and false negative predictions. Among many available programs for predicting mRNA targets for specific miRNA, none of these programs can be used as an independently approach for validating the targets, and all predicted targets must be validated
2.2. Brief introduction to lncRNA
LncRNAs are those longer than 200 nt, and many of them can also act as primary transcripts for the production of short RNAs . It is estimated that total number of lncRNA transcripts, including new unexplored, is approx. 15000. Thousands of protein-coding genes in humans harbour natural antisense transcripts (approx. 61 % of transcribed regions show antisense transcription) belonging to the lncRNA, and majority of known lncRNAs in some way overlap protein-coding loci. All these data are giving the importance to lncRNA annotation .
LncRNAs can be classified according to their proximity to protein coding genes. There are five categories of lncRNAs: sense, antisense, bidirectional, intronic, intergenic. Just to mention a few of them, lincRNAs, a class of ncRNAs, exhibit a high conservation between different species; they both up- and down- regulate hundreds of gene expression and participate in the establishment of cell type-specific epigenetic states . Further, ncRNAs were found expressed at enhancer regions, suggesting that some enhancer RNA is also transcribed with an average size of 800 nt; these transcripts are termed eRNAs. Studies propose a possible role for eRNAs as transcriptional activators, however, question remains whether such eRNAs are in fact a subset of the activating lncRNAs. Similar to eRNA, a novel diverse class of ncRNAs has been linked to the promoters, called PARs, ranging from 16-36 nt to 200 nt. It is suggested that they participate in the transcriptional regulation . Most lncRNAs are characterized by low expression levels, low level of sequence conservation, by composition of poly-A tail and without poly-A tail as well as by spliced and un-spliced forms. They are believed to have nuclear localization, but can also accumulate in cytoplasm of cells .
lncRNAs may act through diverse molecular mechanisms, and play regulatory as well as structural roles in different biological processes . Many of the identified lncRNAs show spatial- and temporal-specific patterns of expression. Almost every step in the life cycle of genes – transcription, mRNAs splicing, RNA decay, and translation – can be influenced by lncRNAs. Generally lncRNAs have been implicated in gene-regulatory roles, such as chromatin dosage-compensation, imprinting, epigenetic regulation, cell cycle control, nuclear and cytoplasmic trafficking, cell differentiation etc. . A number of studies suggest that lncRNAs are key components of the epigenetic regulatory network . Two general modes of lncRNAs regulation seem to be important: interaction with chromatin remodelling complexes that promote silencing of specific genes; and modulation of splicing factors. Chromatin remodelling guided by ncRNAs contributes to the establishment of chromatin structure and to the maintenance of epigenetic memory. Various ncRNAs have been identified as regulators of chromatin structure and gene expression . Additional mechanisms of action are yet to be revealed .
The lncRNA database provides sequence, structural, and conservation evidence for multi-species lncRNAs, together with a list of lncRNAs that are experimentally known to interact with coding mRNAs, harbouring other short ncRNAs and other characteristics of specific lncRNA .
3. Involvement of ncRNA in cancer
Three major mechanisms are known to give rise to deregulated ncRNAs function, genetic alterations, epigenetic alterations, and in case of miRNAs, an aberrant miRNA biogenesis machinery. Since brief overview of first two mechanisms is described below, will be here mentioned only aberrant machinery of miRNA processing. Proteins involved in miRNA biogenesis (Drosha, Dicer, Ago) are deregulated in several cancers. Co-factors involved in miRNA biogenesis can be mutated causing consequently deregulation of Dicer; Exportin 5, mediating pre-miRNA nuclear export, is often mutated and truncated, leaving pre-miRNAs within nucleus [31,32].
3.1. Mutations, SNPs and epigenetics of ncRNAs
Cancer cells have different genetic and epigenetic changes from their normal counterparts and the role of ncRNAs in mediating these differences is beginning to emerge. Specific genetic polymorphisms are associated with the risk of developing several types of cancer [7-9]. Multiple studies have identified small-scale and large-scale mutations and genomic alterations affecting also noncoding regions of the genome. Some of these mutations are structural alterations, rearrangements and chromosomal translocation, amplification, loss of heterozigocity and copy-number variation, nucleotide expansion, and single-nucleotide polymorphisms (SNPs), and they are linking distinct types of mutations in ncRNA genes with diverse diseases . First, lncRNA have already been implicated in human diseases such as cancer and neurodegeneration . Second, approx. half of miRNA genes are encoded in genomic region prone to cancer-associated rearrangements or in fragile chromosomal sites (amplified, deleted or rearranged) that are often associated with cancer, such as ovarian and breast carcinomas, and melanomas [8,11]. Third, presence of SNPs in miRNAs, where disruption of miRNA target interaction either in the miRNA gene or its target site (3′-UTR mRNA) can lead to complete gain or loss of the miRNA function or target gene thus causing disease [34,35]. In contrast to the miRNA target sites in mRNA transcripts, where the potential of variation is huge, variants identified in miRNA precursor sequences tend to be rarer . The presence of SNPs in
3.2. Promising role of ncRNAs in cancer: As cancer-subtype classifiers and detection in body fluids
ncRNAs have been recognized as gene-specific regulators. They are similar in activity to a large number of protein transcription factors that are known to be critical in the transformation of cells to a malignant state. Majority of research has been involved in defining the role of miRNAs in cancer; however, lincRNAs have been shown to play role in tumour development by promoting the expression of genes involved in metastasis and angiogenesis . Genome-wide analyses have shown that ncRNAs have distinct signatures specific for a certain cancer type. Importance of combining ncRNAs with other biomarkers for cancer detection and prognosis would improve cancer risk assessment, detection, and prognosis. Thus, there is a need to combine genomic mutations with ncRNA markers to develop marker panels for more accurate risk assessment and early diagnosis [7-9].
Most cancers are diagnosed in advance stages, leading to poor outcome. Intense investigation is going on seeking specific molecular changes that are able to identify patients with early cancer or precursor lesions . Genome-wide expression profiling has examined miRNAs in preneoplasia or their usefulness to predict progression from preneoplasia to cancer. Several lines of evidence suggest the potential usefulness of ncRNAs, particularly miRNAs: first, as signature of early events in carcinogenesis and as biomarkers in early cancer detection, second, as differential indicators of benign tumours, preneoplasia, and neoplasia, and third, that miRNAs and perhaps other ncRNAs might be useful in determining which preneoplastic lesions are likely to progress to cancer. Distinguishing benign diseases and certain non-precancerous lesions from precancerous lesions and metastatic tumours would improve patient outcomes, survival, and reduce patient discomfort . Characterization of ncRNAs involved in the development or maintenance of oncogenic states may therefore define ncRNAs as early biomarkers for the emergence of cancer, and could have have an impact on the development of tools for disease diagnosis and treatment .
miRNAs are believed to be promising potential biomarkers for cancer diagnosis, prognosis and targets for therapy. As potential markers for diagnosis are better classification factors than mRNAs. miRNAs seems to be evolutionarily selected gene regulatory molecules, their expression profiles might therefore be rich in gene regulatory information. Only small percentage of the 16000 genes on the mRNA-expression arrays are regulatory molecules. This difference may be responsible for more efficient microRNA expression arrays in classifying cancer than mRNA-expression arrays [21,45,46]. Some of the key features of miRNAs that make them useful as potential biomarkers can be briefly summarized. First, expression patterns of miRNAs in human cancers appear to be tissue specific. Second, miRNA profiles appear to reflect developmental lineage and differentiation state of the tumours. Third, miRNAs can successfully classify poorly differentiated tumours with high accuracy (~70 %). In contrast, mRNA profiles in the same set of specimens had an accuracy of only 6 %. Therefore, a combination of both miRNA and mRNA profiling data has the potential of enhancing accuracy of tumour classification. Forth, miRNAs can also be profiled and quantitatively measured in formalin-fixed paraffin-embedded tissues. And last, miRNAs are stable in human body fluids of plasma and serum and can be quantitatively measured in microliter quantities of human sera or plasma using qPCR. [45-47].
Highly stable cell-free circulating nucleic acid (cfCNA), both RNA and DNA, has been discovered in the blood, plasma, and urine in humans. Since there is good correlation between tumours and genetic, epigenetic and/or transcriptomic changes and alterations in cfCNA levels, it gives a usefulness of cfCNA as biomarkers for clinical applications. Release of cfCNA in body fluids is probably related to apoptosis and necrosis. Circulating RNAs are stable in serum and plasma in spite of high amounts of RNAase in blood of cancer patients . They are packed in microparticles, of which the most analyzed are in recent years exosomes [3,49,50]. Tumour derived exosomes are small membrane vesicles of endocytic origin released by the tumour and found in peripheral circulation. Several recent reports showed that exosomes could be an important resource of cf-lncRNA/cf-miRNA in serum or plasma . Small size, relative stability and resistance to RNAase degradation make the miRNAs more superior molecular markers than mRNAs . Using non-invasive diagnostic procedures, the extraction and reliable determination of cf-miRNAs, circulating in body fluids like plasma, serum, and others, could serve as circulating tumour biomarkers [52,53].
LncRNAs show greater tissue specificity compared to protein-coding mRNAs, making them attractive in the search of novel diagnostics and/or prognostics cancer biomarkers in body fluid samples. For an example, lncRNA PCA3 was initially identified as over-expressed in prostate tumours relative to benign prostate hyperplasia and normal epithelium. It was latter showed that is very specific prostate cancer gene, whose mechanism is not yet identified, but it can be detected in urine samples and has been shown to improve diagnosis of prostate cancer .
3.3. ncRNAs can act as both tumour suppressor genes and oncogenes
There are different ways in which miRNAs appear to be involved in cancer: as tumour suppressors, as oncogenes, or as agents involved in affecting genome stability. Below is discussed role of miRNAs acting both, as tumours suppressor and as oncogenes, since are much more investigated in this field than are lncRNAs. Care must be taken in assigning oncogenic or tumour suppressor activity to a miRNA, since miRNA expression patterns are highly specific for cell-type and cellular differentiation status. The same miRNA can function as tumour suppressor in one cell type and as potential oncogene in other cell type. Some of the aberrant miRNA expression observed in tumours may also be a secondary consequence of the loss of normal cellular function that accompanies malignant transformation. Up- or down-regulation of a miRNA in a given tumour type is not obvious a causative role in tumorigenesis .
The increased expression of oncogenic miRNAs appears to act in a manner analogous to an oncogene. Over-expression of oncogenic miRNAs are presumed to function by down-regulating the levels of protein product of target tumour suppressor gene or by reduction of tumour suppressor processes, such as apoptosis [2,6,20]. A loss of expression of tumour suppressor miRNA may lead to elevated levels of the protein products of target oncogenes , activation of an oncogenic processes, such as proliferation [2,20]. MicroRNAs with anti-proliferative and pro-apoptotic activity are likely to function as tumour suppressors and thus may be under-expressed in cancer cells. Figure 1 represents schematic overview of miRNAs acting as tumour suppressors or oncogenes in comparison to non-cancerous cells.
There should be at least four type of evidence before assigning tumour suppressor function to ncRNAs: (i) data about widespread deregulation in diverse cancer, (ii) gain or loss of function in tumours owing to deletion, amplification or mutation, (iii) direct documentation of tumour suppressing activity using cell line or animal models, (iv) the identification and verification of cancer relevant targets that define mechanisms through which miRNAs participate in oncogenesis .
4. ncRNAs as potential therapeutic targets in cancer
4.1. RNAi in therapeutic applications
Using RNAi approaches, ncRNAs may in future serve as therapeutic targets. For ncRNA that is under-expressed and possess tumour suppressor function, re-introduction of the mature ncRNA into the affected tissue would restore the regulation of the target gene. By contrast, over-expressed ncRNA with oncogenic function could be down-regulated by reducing mature ncRNA level by its direct targeting .
Due to the interferon response it is difficult to introduce long dsRNAs into mammalian cells, however, the use of RNAi as a therapeutic approach has been successfully used. Among the first applications to reach clinical trials were in the treatment of macular degeneration and respiratory syncytical virus infection, reversal of induced liver failure in mouse models, antiviral therapies, neurodegenerative diseases, and cancer. Cancer was treated by silencing up-regulated genes in tumour cells or genes involved in cell division. A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method, which to date has involved mainly viral (lentivirus, adenovirus, adeno-associated virus) and non-viral (nanoparticles, aptamers, stable nucleic-acid-lipid particle, e.g.) vector systems similar to those suggested for gene therapy .
4.2. ncRNAs with tumour suppressor function as therapeutic targets
Pharmacological manipulation of miRNAs is still in its infancy; however, the correlation between the expression of miRNAs and their effects on target oncogenes, on tumorigenesis, and on the proliferation of cancer cells has gained experimental support. miRNAs are small molecules, making their
For miRNA that is under-expressed, re-introduction of the mature miRNA into the affected tissue would restore regulation of the target gene. For this purpose, artificial miRNA (miRNA-mimic) have been developed to enhance the expression of beneficial miRNAs or the introduction of short hairpin duplex, similar to
miRNA mimic can only last a couple of days and the long term biological effects were not observed very effectively. To overcome this, the cells were infected with a lentivirus that expressed mature miRNAs. This generated stable cell expressing miRNAs. miRNA mimics and lentiviral miRNAs showed great potential in restoring tumour suppressor miRNAs. However, viral and non-viral delivery systems have been developed. Viral vector-directed methods show high gene transfer efficiency, but have some limitations. However, non-viral gene transfer vectors have been also developed: cationic liposome mediated gene transfer system, lipoplexes, neutral lipid emulsion, etc. .
Expression of miRNA-mimic would simultaneously suppress many gene targets. miRNAs-mimic would be useful in conjunction with standard chemotherapy or radiotherapy, by influencing drug resistance or enhancing responsiveness to therapy. Current limitation is need for improvement of efficiency of delivery to target tissue, for systemic drug administration, potential inhibition of non-target genes (“off-target effect”), redundancy among miRNAs efficacy, potential toxicity and immunogeneic responses. However, studies introducing miRNAs strategies to inhibit cancer propagation in animal models are showing promising results .
Therapeutic delivery to animal models was demonstrated using miRNA-mimics of the tumour suppressor miRNAs,
Successful inhibition of lncRNAs seems to be more difficult than inhibition of miRNAs. Our growing knowledge of other ncRNAs might exploit in future to develop new therapeutic strategies not only against cancer, but also for other diseased states. The findings regarding lncRNA and Alzheimer disease are attracting the attention of pharmaceutical and biotechnology industries . Therapy using small RNAs that targets ncRNA transcripts, such as eRNAs or PARs, may represent a new way to treat disease conditions caused by epigenetic changes .
Another possible approach for manipulation of ncRNAs level may also be by altering DNA methylation. As mentioned above, DNA methylation is a crucial mechanism associated with epigenetic regulation. It has been shown that in cancer cells treated with DNA demethylating agent reactivation of certain miRNAs occurs . ncRNAs mediated therapy may also be useful in combination with DNA methyltransferase inhibitors that are other way toxic .
5. ncRNAs as tumour suppressor in different types of cancers
5.1. miRNAs as tumour suppressors
In the following section, down-regulated miRNAs will be describe and miRNAs with suggested tumours suppressive roles in different types of cancer. However, down-regulation does not ncessary mean that miRNA is tumours uppressor.
Breast cancer is one of the most important cancers in adult females.
Colorectal cancer is the third most commonly diagnosed cancer in the world, but it is more common in developed countries. Also in colorectal neoplasia miRNAs expression is associated to the tumour formation. Reduced expression of
Aberrant DNA methylation may further induce silencing of specific miRNAs in CRC. While methylation of
Gastric cancer is the fourth most common cancer and the second leading cause of cancer death in the world. It was reported that loss of Ago2, which leads to premature stopping of miRNAs biogenesis and general deregulation of miRNAs expression, was observed in 40 % of human gastric cancer patients with high microsatellite instability. A number of miRNAs were reported to be down-regulated. Among these,
An epigenetic silencing of
The association between genetic polymorphism of
Pancreatic cancer is the eighth most common cause of cancer-related deaths worldwide and it has a poor prognosis for all stages. It is usually diagnosed at advent stages, therefore it is an urgent need to find some specific biomarkers and key components of carcinogenesis. Several miRNAs were reported to suppress metastasis. In pancreatic cancer cell lines,
Primary liver cancer mainly refers to HCC, which is one of the most common malignant tumours in liver and accounts for 85-90 % of primary liver cancers. Cyclins D2 and E2 were validated as direct targets for
Lung cancer is one of the most common cancers of adults and is also leading cause of cancer-related deaths in many economically developed countries .
Mutations in EGFR gene are more frequent in NSCLC patients who never smoked tobacco; a significant down-regulation of
It was proposed that
The phrase “brain tumours” describes an inhomogeneous collection of various tumours of the brain, which represents primary tumours of nervous central system or metastases. Glioblastomas (belongs to family of gliomas) are the most frequent occurrence and malignant form of primary brain tumors in contrary to medulloblastomas, which have a better prognosis [2,11]. Several articles have described the effects of ectopic miRNA modulation on medulloblastoma cell proliferation and growth.
Rescued expression of
In contrary to medulloblastomas, are gliomas the most common and deadly primary human brain tumours, and its subtype glioblastomas are highly invasive, very aggressive, and one of the most incurable .
Head and neck tumours are a heterogenous group with different behaviour at the various sites arising from anatomical factors, cell-type variation, and differences in exposure to risk factors including tobacco, alchocol, and viruses . Head and neck squamous cell carcinoma is represented by epithelial cancers of the oral cavity, pharynx, nasal cavity, paranasal sinuses, salivary glands and larynx . Studies were made in expression profiling regarding different sites of head and neck tumours, tongue, tonsil, larynx, hypopharynx, nasopharynx, saliva, oral cavity, salivary gland and animal models. Several miRNAs were identified as down-regulated, and for some of their target genes were validated .
Low expression of
Deregulation of miRNAs in cancer can occur through epigenetic changes (promoter CpG island hyper-methylation in the case of
Melanoma is the most aggressive type of skin cancer, and it is resistant to therapy in its advanced stages . Abnormalities in several signal transduction pathways, which are important for normal melanocyte development, only partly explain molecular mechanism directly linking UV radiation to the development of melanoma. miRNAs are emerging as important causal factors to melanoma initiation and progression . In 45 primary cultured melanoma cell lines, there was observed that many genomic loci containing miRNAs are frequently affected (85.9 %) by copy number abnormalities. For an example, copy number losses of the region containing
However, deregulation of miRNAs expression is not always explained.
5.2. lncRNAs as tumour suppressors
lncRNAs are known to mediate epigenetic modifications of DNA by recruiting chromatin complexes to specific loci . Only a handful of lncRNAs have been characterized, and their involvement in control of gene expression . We therefore presented four lncRNAs with proposed tumour suppressor function in cancer.
It is involved in the regulation of Cyclin D1 gene expression. Cyclin D1 is a cell cycle regulator often mutated, amplified and over-expressed in various types of cancer. After binding of this lncRNA on RNA-binding protein, consequently inhibition of enzymatic activities of the histone acetyltransferases occurs, leading to silencing of cyclin D1 gene. These studies suggest that this lncRNA is a tumour suppressor RNA, which can be rapidly induced by cellular stress to regulate it sense gene expression .
The rest of ncRNAs, other than miRNAs, in regulation biological functions are more or less unexplored, and this should be further investigated in future research. Regarding therapeutic approaches, we still need more knowledge concerning which miRNAs to target, how to produce and stabilize them, how to direct them to the target tissue. The specificity of drug-like oligonucleotides is important, because of the off-target effect. The off-target effect is also a significant challenge, especially considering that miRNA-mediated repression often requires a homology of only six to seven nucleotides in the seed region of the miRNA and mRNA target site. Toxicity due to chemical modifications, which is used to facilitate cellular uptake and prevent degradation, should be take into account. However, only recently was described the possibility of using exososmes and exosomal tumour-suppressive miRNAs as novel cancer therapy .