ReviewNon-coding RNAs as a new dawn in tumor diagnosis
Introduction
In the last years it has become increasingly clear that the mammalian transcriptome is extremely complex due to the inclusion of a large number of small non-coding RNAs (sncRNAs) and long noncoding RNAs (lncRNAs). Non-coding RNAs (ncRNAs) are a heterogeneous class of transcribed RNA molecules from non-(protein)-coding regions, which lack an open reading frame and consequently have no apparent protein-coding ability. Based on the size of the functional RNA molecule, regulatory ncRNAs are classified analytically as long ncRNAs (lncRNAs, >200 nt), and small ncRNAs (sncRNAs, 18–200 nt) [1], [2], [3].
RNA biomolecules have been identified since the late 1800s, but their fundamental roles in cell functions have long been overshadowed by DNA and proteins. From 1950s, with the clarification of the molecular structure of DNA, it was proposed that RNA would be an intermediate molecule in the flow of genetic information from DNA to proteins, as assured in the central dogma of molecular biology. On the basis of the accepted importance of proteins in exerting biological functions, RNAs have been regarded for a long time as merely mediators in passing genetic codes to final protein molecules. As a consequence, the functional activities of RNAs themselves were largely neglected. Ribosome RNAs (rRNA) and transfer RNAs (tRNA) are among the early-discovered non-coding RNA transcripts. However, given their roles in facilitating protein translation, they are still considered part of the machinery translating genomic code into protein synthesis. Nonetheless, the discovery of these ncRNAs opened the field of regulatory RNAs with no or little protein-coding potential. Since then many new classes of regulatory non-coding RNAs have been identified and remarkable progresses have been made in elucidating their expression, biogenesis, mechanisms and function in many, if not all, biological processes, including cancer development and progression [4], [5].
The idea that RNAs are much more than molecules involved in storage/transfer of information emerged since the discovery of ribozymes, endowed with active roles as catalysts of chemical reactions in cells. Indeed, RNA has been suggested to be the earliest molecule of life and thought to possess both informational and catalytic function [6], [7], [8]. These discoveries clearly encouraged a variety of studies to search for potential new roles of RNA molecules in vivo, and led to the re-evaluation of RNAs as crucial molecules in the evolution of life. Current advances on the sequencing technologies revealed that vast majority of the human genome is transcribed, while the protein-coding genes occupy only about 3% of the human genome [9]. Therefore, the widespread transcription of the genome into non-protein-coding RNAs strongly imply that RNAs are capable of process functions other than mere mediators between DNA and proteins, and emerging evidences from the last two decades have unambiguously proved the functional importance of non-coding RNAs in human biology and diseases. As a result, the involvement of RNAs in other critical molecular processes in eukaryotic cells was progressively revealed, as in the case of DNA replication, protein translation and RNA transcript maturation. With time, many small non-coding RNA molecules were isolated and characterized; among the first, U1, U2, U4, U5 and U6 small nuclear RNA (snRNA) as fundamental parts of ribonucleoproteic complexes (RNP), later identified as the components of the splicesome [10]. Thereafter, it was showed that RNA editing mechanisms, based on protein or protein-RNA complexes and regulating the information content of tRNA, rRNA and mRNA molecules, require a “guide RNA” molecule, which, through base-pairing with the target RNA molecule, determines the editing site [11]. In addition, post-transcriptional processing and modifications of rRNAs, essential for the production of efficient ribosomes, is directed by two large guide families of small nucleolar RNAs (snoRNA) [12].
In the wake of these findings, in the mid-1980s Blackburn and Greider came to the discovery of telomerase by demonstrating the existence of an enzymatic activity within cell extracts inserting tandem hexanucleotides to chromosome ends [13], [14]. More recently it was hypothesized that telomerase arise by the association of an ancient ribozyme with the reverse-transcriptase subunit, showing a mechanism resembling that of pure ribozymes, and placing the telomerase as a missing link in the evolution from RNA enzymes to protein enzymes [15].
Already at this point, the growing descriptions of the importance of RNA molecules for cell life started to push them to public and scientific interest, but the complexity of their roles and the wide multiplicity of molecular mechanisms in which RNA molecules are critical players was still far from clear. The discovery of microRNAs (miRNAs) in early 1990s opened a new chapter of gene regulation by non-coding RNAs and represented a crucial boost for investigations on the RNA molecules not coding for proteins [16]. Concurrently, the observations that exogenously introduced double stranded RNA (dsRNA) and plasmids expressing short hair-pin RNA (shRNA) specifically base-pairing with target mRNA molecules were able to trigger mRNA degradation (RNA interference, RNAi) revealed, for the first time, that specific silencing pathways based on sncRNAs are operating in eukaryotic cells [17], [18]. These observations led to the development of the RNA interference (RNAi) technique that has been extensively used in the study of gene function [19], [20].
As mentioned before, in recent years the use of genome wide approaches and the large output of genome sequencing technologies have revealed that the mammalian transcriptome is much more complex than previously hypothesized, since it includes a large number of small non-coding RNAs (sncRNAs) and long non-coding RNAs (lncRNAs) [21], [22]. miRNAs are an abundant class of endogenous non-coding small RNA molecules, 20÷25 nucleotides in length, which act as either oncogenes or tumor suppressors genes and thus have crucial roles in carcinogenesis [23], [24]. Different other types of small non-coding RNAs have also been subsequently identified, including endogenous small interfering RNAs (endo-siRNAs), PIWI-associated small RNAs (piRNAs), small nucleolar RNAs (snoRNAs), sno-derived RNAs (sdRNAs), transcription initiation RNAs (tiRNAs), miRNA-offset RNAs (moRNAs), and others [25]. Conversely, the lncRNAs family contains multiple classes of RNAs, which are nuclear RNAs transcripts longer than 200 nucleotides, involved in the regulation of cellular processes such as apoptosis, proliferation and metastases development, thereby emerging as important regulators in a wide range of biological activities and human diseases [26]. Currently, a systematic classification of long non-coding RNA is missing; nevertheless, according to their genomic localization or other biological features, they are classified as natural antisense transcripts, long intergenic non-coding RNAs, transcribed ultraconserved regions (T-UCRs), circular RNAs, enhancer-associated RNAs, promoter-associated RNAs, and others [27]. Similar to miRNAs, the dysregulation of lncRNAs is associated with many human cancers and defines their phenotypes [28]. The expression profiling of both lncRNA and miRNA is deeply different depending upon both the histological type of the tissues and pathologic/not pathologic conditions as their expression is different in cancer tissues if compared to normal counterparts. Therefore, lncRNAs and sncRNAs, by means of their ability in post-transcriptional regulation of gene expression and in target gene translation, may become useful non-invasive diagnostic biomarkers and powerful tools in cancer prevention and treatment. Indeed, several emerging evidences have revealed for both lncRNAs and sncRNAs a close correlation with cancer development and progression, so that some ncRNAs have already been used as biomarkers and targets in cancer management, for diagnosis and targeted therapy, respectively [29]. Hence, the discovery of the biological functions related to ncRNAs have undoubtedly animated the scientific community and stimulated biomedical studies that are nowadays changing the way for cancer diagnosis and treatment.
Section snippets
Biogenesis and functional role of non-coding RNAs
The paradox that less than about 2% of the total human genome is recruited for protein expression, can be partly explained by the increase in diversity and functionality of the human proteome achieved through alternative pre-mRNA splicing, as well as through post-translational modifications of proteins [30]. In recent years, it has increasingly become more evident that the non protein-coding portion of the genome is of critical functional relevance in several mechanisms of gene regulation, both
Mechanism and clinical relevance of exosome-mediated miRNA secretion
About 15 years after the first identification of miRNAs, it has been discovered that they were detectable in body fluids encapsulated in lipid microvesicles [57]. Extracellular vesicles (EVs), characterized according to the size into exosomes (<100 nm), microvesicles (1000 nm) and apoptotic bodies (1–4 μm) [58], originate from cells and are able of transferring miRNAs, mRNAs and proteins in both paracrine (connecting cells belonging to the same tissue) and endocrine (to distant target cells)
ncRNA dysregulation in cancer: preclinical studies and therapeutic implications
Recent studies indicate that miRNAs are deregulated in many types of tumors, being involved in several cancer processes, such as cell proliferation, invasion and metastasis. miRNAs can, in fact, inhibit the expression of genes involved in many cellular pathways regulating crucial mechanisms like cell cycle, cell death and cell migration. Numerous experiments and clinic analyses suggest that miRNAs may function as a novel class of oncogenes (oncomirs) or tumor suppressor genes. Oncomirs are
Potential of ncRNAs as novel biomarkers for solid and hematologic malignancies
Improved knowledge of ncRNAs’ expression pattern and function may lead to a better understanding of the heterogeneity of malignancies and, most likely, also lead to their use as diagnostic, prognostic and therapeutic targets. The best-studied ncRNAs category is represented by miRNAs, which have emerged as suitable diagnostic and prognostic biomarkers with the capacity to drive treatment decisions in the clinical setting. Researchers have identified miRNA signatures in serum, plasma, peripheral
miRNA detection and quantification in body fluids
miRNAs, unlike other biomarkers, are highly stable and can be isolated from tumor tissue samples after formalin and paraffin passages, and from serum and plasma samples after being at room temperature for 24 h and after freezing and de-freezing cycles [189]. In addition, miRNAs appear to be resistant to RNAses present in the plasma probably due to their small size or molecular structure, as well as in virtue of their loading in EV. The presence of circulating miRNAs has been demonstrated in
Challenges, opportunities and pitfalls of miRNA profiling
Tissue biopsies are still considered the gold standard for molecular evaluation of cancer, although this procedure is invasive and has several limits related to the low possibility of sampling and to the heterogeneity of tissues, the reason why biopsy only gives a small sampling of the entire tumor. These limitations account for the potentially missing clinical information coming from bioptic tissues. Moreover, another severe restriction is represented by the little opportunity to gain multiple
Conclusions
The remarkable importance of an early cancer diagnosis, monitoring and treatment, in view of an efficient patient’s management, has provided a strong boost to the recognition of tumor-specific, non invasive, and easy to detect and quantify biomarkers. Both blood-based and tissue biopsies-obtained protein markers, until now considered the gold standard for molecular evaluation of cancer, have shown limited specificity and sensitivity. In this review we have focused on the possibility to
Conflict of interest
None.
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These authors contributed equally to this work.