microRNA: Small RNA molecules of great utility as diagnostic biomarkers in lung cancer

What scientists didn’t know then was that, as soon as 10 years later, these unsuspected small RNA molecules, dubbed ‘microRNAs’ (miRNAs), would already have been found in organisms as evolutionarily diverse as man, worm, Drosophila, and also in the small plant Arabidopsis thaliana⁴. These findings represented a paradigm shift in our understanding of the mechanisms regulating gene expression⁵. Today, more than 25,000 miRNAs have been described in 193 different species, and more than 2,000 miRNAs are known in man⁶.

miRNAs

miRNAs are small noncoding RNAs (∼23 nt) that regulate gene expression at the posttranscriptional level⁷. They derive from endogenous transcripts that fold back on them – selves to form distinctive hairpin structures, which are then processed by dedicated endonucleases (Drosha and Dicer). Once in the cytoplasm, they are incorporated into a ribonucleoprotein complex miRISC, in association with which they can target specific mRNAs in a miRNA sequence-dependent process. By base-pairing to the mRNAs of protein-coding genes, they direct their degradation and translational repression via several mechanisms. Several miRNAs might target a single mRNA, and a single miRNA species might bind to several mRNA species, thus giving rise to a complex gene expression network, whose details, albeit under intensive investigation worldwide, are still far from being understood. miRNAs are often transcribed and processed in a cell type- and developmental stage-specific fashion, and they have been demonstrated to contribute to tissue-specificity of mRNA expression in many human tissues⁸. Given their role in the fine regulation of several cellular processes such as development, differentiation, cell proliferation and apoptosis, it was of no surprise that miRNAs could be implicated in many human diseases, including cancers.

miRNAs and cancer

In a pioneering paper published in 2002, the Croce laboratory showed that the genes for miR15 and miR16 are deleted or downregulated in the majority of chronic lymphocytic leukaemia cases⁹. Since then, evidence has accumulated to indicate that miRNAs play a role in the onset and progression of human cancers¹⁰. The transcription or processing of some miRNAs is altered in neoplastic tissues, in respect to their normal counterparts. miRNAs whose levels increase in tumours are referred to as oncogenic miRNAs (‘onco-miRs’), sometimes even if there is no evidence for their causative role in tumorigenesis. On the other hand, miRNAs down-regulated in cancer are considered tumour suppressors. From the mechanistic point of view, it is important to understand how these variations may contribute to tumour progression.

miRNA dysregulation can be used as a diagnostic tool, even if no regulatory function has been demonstrated for the particular miRNA. In 2005, Lu and colleagues demonstrated the potential for miRNAs as diagnostic tumour markers¹¹, when they were able to indicate the tumour embryonic origin using miRNA expression profiles, successfully classifying poorly differentiated tumours. Indeed in the last seven years, alteration of miRNA levels and/or correlation of miRNA expression with clinical parameters (such as disease progression or therapy response), have been shown in several cancers, indicating that miRNAs can serve as clinically relevant bio – markers (reviewed in¹²).

The advantage of the use of miRNAs as biomarkers resides in the ease of their detection and in their extreme specificity. Furthermore, miRNAs are remarkably stable molecules that have been shown to be well preserved in formalin fixed, paraffin embedded tissues (FFPE) as well as in fresh snap-frozen specimens, unlike larger RNA molecules as mRNA¹³.

The tiny size of these RNA molecules, originally contributing to their obscurity, turned out to be an advantage, when it came to their stability and the specificity of their detection.

Methods for the isolation and profiling of miRNAs

A range of methods have been used for the isolation and profiling of miRNAs. Purification of total RNA is obtained either through several commercially available column filtration protocols, implemented to guarantee recovery of miRNAs, or via the extraction of RNA by variously named ‘Tri-reagents’ (acid phenol in combination with guanidinium-thiocyanate and chloroform), also available from vendors. Given that the interest is focused on the quantification of specific miRNAs in different conditions, the method of choice should exclude any bias in the purification of miRNAs from the samples. Importantly, the Kim laboratory has recently reported that, differently from what everybody in the field has thought for decades, in Trireagents- based RNA purification protocols, short structured miRNAs with low GC content are lost when a small number of cells are used¹⁴. The finding raises warning flags about comparisons of miRNA levels between populations of cells at different densities.

The main methods currently used for miRNA profiling are sequencing, microarray and real-time RT-PCR- based approaches. While next generation sequencing methodologies are rapidly evolving in power and multiplexing capacities and their cost is progressively decreasing, they are just beginning to be used to define miRNA signatures characteristic of specific cancer types or stages. The majority of miRNA profiling studies have been carried out so far using microarrays and have provided signatures consisting in few to several (5-30) distinct miRNAs¹². With time, it has become clear in the field that the use of microarrays for miRNA profiling presents with major problems of cross-hybridisation between members of miRNA sequence families and discrepancies in comparing results obtained with different microarray platforms. A common strategy is to validate the microarray data by qRT-PCR, and several methods for the analysis of miRNAs by qPCR have been devised (reviewed in¹⁵). In fact, several companies are providing multiwellplate- based qRT-PCR assays that promise to substitute microarrays in the high-throughput profiling of miRNAs. Additionally, qRT-PCR is presently the method of choice when it comes to measuring the levels of the restricted number of miRNA biomarkers in cancer samples, in the clinical setting.

miRNA as lung cancer biomarkers

As said, miRNAs have been widely studied in several cancer types. We focused in particular on lung cancer. Lung cancer is the leading cause of cancer mortality worldwide, and 80 per cent of lung cancers are non-small cell lung cancers (NSCLCs)¹⁶. The cause of this high mortality is due to the poor prognosis of this disease caused by a late disease presentation, tumour heterogeneities within histological subtypes and the relatively limited understanding of tumour biology. In this context, the pattern of miRNAs expression profiles could be useful in improving the classification of lung cancers and predicting their behaviour.

With the emergence of targeted therapies directed against specific cellular alterations, an accurate classification of tumours into squamous cell carcinomas (SQCCs) and adenocarcinomas (ADCs) became necessary. Recent studies have shown that not only can miRNAs be used to sub-classify NSCLCs¹⁷ but specific miRNA profiles may also predict prognosis and disease recurrence in early-stage NSCLCs^18-21. However, this can be challenging, especially in cases in which biopsy/aspirate specimens are small or tumours are poorly differentiated. The relative quantification of miR- 205 and miR-21 was reported by us and others to be a useful marker for differentiating SQCCs from non-SQCCs NSCLCs, with a sensitivity of 96 per cent and specificity of 90 per cent, even in small biopsies from poorly differentiated tumours^17,22,23. qRT-PCR data show that miR-21 relative levels are similar in SQCCs and ADCs, whereas miR-205 relative levels are lower in ADCs23. Subsequently, we reported that the quantitative analysis of these miRNAs allows the classification of lung tumours not otherwise classified under the traditional immuno – histochemical methods²⁴. Landi et al also reported a five-miRNA signature (miR-25, miR-34c-5p, miR-191, let-7e, and miR-34a) that accurately differentiates SQCCs from ADCs25. Additionally, the lower expression level of this signature correlated with poor overall survival among SQCCs patients²⁵.

Circulating miRNAs

An early diagnosis of cancer remains a compelling challenge and, in this context, it is important to find a sensitive, non-invasive tool to detect early neoplastic changes. The exceptional stability of miRNAs in several tissues stimulated efforts aimed at establishing if these tiny molecules were also preserved, detectable and quantifiable in the circulation and in other biofluids. From the first publications about miRNAs as blood-based biomarkers for cancer²⁶, miRNAs have been discovered in a host of biofluids (e.g. urine, saliva, CSF and amniotic fluid). miRNAs detected in biofluids may have a cellular or an extracellular origin, the latter being the most interesting in terms of biomarker discovery. Circulating or extracellular miRNAs display remarkable stability²⁷ and resistance to degradation from endogenous RNase activity²⁸, by inclusion in various protein complexes or membranous particles such as exosomes or microvesicles²⁹. It has also been shown that miRNAs present in body fluids can reflect altered physiological conditions, representing new effective biomarkers³⁰. All these findings suggest that extracellular miRNAs may have biological functions akin to that of signalling molecules and hormones. The ease and reliability of determining body fluid miRNAs profiles paved the way for several applications, both in the research laboratory and in the clinic.

miRNAs studies could open a new era in cancer treatments, providing improved targeted agents for the cure of patients (reviewed in³¹), and forming the basis for the development of novel therapeutics and/or early disease biomarkers.

Acknowledgements

Work in the laboratory of M.A.D. is supported by a Centre for Integrative Biology (University of Trento) start-up grant. Work in the M.B. lab is supported by grants of the Provincia Autonoma di Trento and of the Fondazione Cassa di Risparmio di Trento e Rovereto.

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Biographies

Michela A. Denti obtained her PhD in Biology from the Scuola Normale Superiore in Pisa, Italy, in 1997, followed by a postdoctoral training in RNA biology, in the laboratory of Professor Martin Tabler at the Institute for Molecular Biology and Biotechnology, Heraklion, Greece. In 2001, Dr. Denti started a second post-doc tenure in the laboratory of Professor Irene Bozzoni, at the University La Sapienza in Rome, Italy, where she focused on the applications of RNA interference and on RNA-based therapeutics. In 2008, Dr. Denti obtained an Assistant Professor position at the Centre for Integrative Biology (CIBIO), University of Trento, Italy, where she leads the Laboratory of RNA Biology and Biotechnology, investigating the role of microRNAs in pathogenesis and the therapeutic use of antisense-RNAs to induce exon-skipping.

Margherita Grasso obtained her PhD in Cellular Sciences and Technologies from the University La Sapienza in 2010, followed by a post-doctoral training in Department of Anatomy, Histology, Forensic Medicine and Orthopaedic at University La Sapienza with a research project concerning the development of a transgenic mouse line for the study and characterisation of spermatogonial stem cells. In 2011, Dr. Grasso started a second postdoc in the laboratory of Dr. Denti, where she focuses on the role of microRNAs in cancer and neurodegenerative diseases.

Mattia Barbareschi graduated at the School of Medicine of the Milan State University, Italy, in 1983, and specialised in Anatomic Pathology, at the School of Medicine of the State University of Brescia, in 1989. He was a Postdoctoral Fellow at the third Department of Surgical Pathology, School of Medicine, Milan State University, from 1985 to 1989 and Visiting Clinician at Mayo Clinic Scottsdale in 1999, 2001 and 2003. Since 1990, he has been Associate Director at the Department of Surgical Pathology and Cytopathology of the S.Chiara Hospital in Trento, Italy. Since 2008 he has been Director of the ‘Trentino Biobank’ (an oncological tissue bank) and Director for the Molecular Pathology Laboratory of the Dept. of Surgical Pathology and Cytopathology of the S.Chiara Hospital in Trento (diagnostic molecular pathology aimed at viral diagnosis, LOH, microsatellite instability, gene mutation analysis, FISH, CISH, Her2/neu alterations, etc).

Chiara Cantaloni graduated in Industrial Biotechnology (2004) and in Medical Biology (2011) from the University of Bologna. Since 2008, she has been a fellow at the Surgical Pathology Department, S. Chiara Hospital, Trento, where she has been responsible for the preparation of Tissue Microarrays blocks, for the IHC of gastrointestinal stromal tumours and, more generally, for the biological frozen material of the ‘Trentino Biobank’. Recently, she collaborates with CIBIO also in a project focusing on recurrent genomic alterations as biomarkers predictive of the response to brachytherapy treatment in prostate cancer.