Expression profiling of circulating miRNAs as a novel non-invasive diagnostic tool

microRNAs (miRNAs) are an abundant class of short single stranded non-coding RNAs (~22 nts) that regulate gene expression at the posttranscriptional level. Interaction between an miRNA and any given of its mRNA targets results in either translation inhibition, mRNA degradation or a combination of both mechanisms. Therefore, miRNAs activity effectively reduces the transcriptional output of a target gene, without affecting its transcription rate. Currently, the sequence of over 60,000 microRNAs are deposited in the miRBase database [Version 17, April 20116]. miRNA activity has been associated with the control of a wide range of basic processes such as development, differentiation and metabolism. Detection of differential expression of miRNAs in many cases have established the basis for miRNA functional analysis and specific miRNA expression patterns can provide valuable diagnostic and prognostic indications, for example, in the context of human malignancies^7,8. Moreover, the deregulation of the expression of miRNAs has been shown to contribute to cancer development through various kinds of mechanisms, including deletions, amplification or mutations involving miRNA loci, epigenetic silencing, as well as the dysregulation of transcription factors that target specific miRNAs^9,10.

Circulating miRNA; a novel non-invasive diagnostic tool

Recently, cell-free miRNA have been isolated and measured in the blood¹¹. The mechanism of how cell-free miRNAs are released into the circulation is yet unclear. To date, two different populations of circulating miRNAs have been identified; one associated to vesicles (exosomes and apoptotic bodies) and one associated to the Argonaute protein 2 [Ago2¹²]. Although cellular fragmentation resulting from apoptotic processes is thought to be at the origin of circulating miRNA, increasing evidence suggests that not only exosomes-associated miRNAs can be actively secreted⁹, but also that miRNA-containing microvesicles can be efficiently delivered into recipient cells^13,14. However, whether the levels of circulating miRNAs are able to regulate or coordinate gene expression at endocrine or paracrine level¹⁵ it is a crucial question that needs to be elucidated. The existence of a specific population of circulating miRNAs in the blood of healthy as well as diseased individuals have raised the possibility that changes in miRNA levels may serve as a biomarker for diagnosis, prognosis and therapeutic response of patients. Importantly, these miRNAs are stable, even after multiple freeze-thaw cycles¹6, which is an important prerequisite for utility as a biomarker¹⁷ and can be easily detected by using microarray¹⁸ or quantitative real-time PCR [qPCR¹⁹], and even Next Generation Sequencing [NGS²⁰] approaches.

Purification and quantification of circulating miRNAs

During the past few years, our lab has developed and optimised dedicated protocols for the quantification and analysis of miRNA expression profiling in tissues and cells by using microarray²¹ and qPCR (miQPCR, manuscript in preparation). Furthermore, we have recently shown that the reliability and reproducibility of miRNA profiling are a function of the quality of the RNA used as input material²². Thus, a robust method for RNA isolation is essential. Because of the great interest in detecting circulating miRNAs, new dedicated protocols and products are emerging throughout the literature and in the portfolio of biotech companies (i.e. Exiqon). Technically, there are three major challenges associated to the analysis of circulating miRNAs: i) plasma and serum contain polymerase inhibitors; ii) isolated nucleic acids are too diluted to be easily detected; and iii) no endogenous reference genes are available for normalisation²³. Technical approaches have been developed to solve problems associated to point (i) and (ii). However, the biggest challenge is the lack of reliable reference genes. As a matter of fact, qPCR data of miRNA levels in plasma of patients affected from the same type of tumour but carried out in different studies show conflicting results²⁴. The observed discrepancies are most likely due to the lack of common reference genes. Suggested solutions include using a microarray-like approach by performing a median normalisation of the complete qPCR data²⁵. However, this type of normalisation is feasible only if a large enough number of miRNAs and samples are analysed, leaving small scale projects with the problem of selecting and evaluating universal reference genes. As an alternative, we have developed an approach where carrier RNA, containing yeast RNA and artificial miRNAs, is added to the samples before RNA purification. This approach enables us to estimate the amount of RNA which is recovered (or lost) during RNA extraction. In addition, the employment of the spike-ins controls (i.e. artificial miRNAs) as reference genes allows the assessment and correction for differences in RNA recovery, cDNA synthesis as well as qPCR efficiency due to the presence of polymerase inhibitors.

Analysis of circulating microRNAs by using quantitative real-time PCR

Recently, our group has developed a novel approach to synthesise cDNA that we have named miQPCR (manuscript in preparation). The miQPCR method uniformly elongates all miRNAs contained in the sample by supplying a universal sequence. The extended miRNAs are then converted to cDNA through standard reverse transcription protocols. The advantage of the miQPCR over existing universal elongation approaches (i.e. polyadenylation) is that the addition of the linker extends all the miRNAs within the sample of a specific sequence. This allows both an increased efficiency in cDNA synthesis, as well as enabling flexible miRNA Specific Primers (miSPs) design. In fact, our innovative approach allows the design of temperature adjusted miSP to such an extent that virtually all primers we design have a predicted temperature of 55-60°C (which is the optimal temperature range for qPCR). Importantly, this is achieved without the necessity of modified nucleotide [i.e., LNA²⁶ or ZNA²⁷]. Our current work is focused on adapting the miQPCR approach to suit the detection of circulating miRNA in serum and plasma prepared from human and mouse blood.

Circulating miRNAs are potential biomarker for the detection of human diseases

During the past decade, miRNA activity has been associated with the control of a wide range of processes such as development, differentiation and metabolism²⁸. Furthermore, it has been shown that dysfunctional expression of miRNAs is associated to the development of a number of human diseases, including cancer^9,10. Several studies have identified miRNA expression signatures that classify cancer patients into different prognostic groups and survival prediction^7,8. However, this experimental approach requires the isolation of cancer material through invasive techniques, limiting the effective diagnosis and surveillance of these complex multi-factorial diseases.

With this premise, circulating miRNAs have been ‘tagged’ by the biomedical community as easily accessible biomarkers for the screening of cancer patients. Indeed, during the past two years, several studies have been published. For example, the levels of circulating miRNAs have been reported to be potential diagnostic and prognostic biomarkers for different types of cancer including but not limited to breast cancer^19,29, lung cancer^20,30, prostate cancer³¹ and hepatocellular carcinoma^32,33. In addition, Brase and colleagues³¹ have identified that levels of circulating mir-375 and miR-141 are not only able to positively identify prostate cancer, but also that their relative amount correlated with clinicpathological variables of tumour progression. Furthermore, the utility of circulating miRNAs as biomarkers is not limited to cancer, and although cancer studies are the ones making the headlines, different types of diseases can be assessed by using this methodological approach. In fact, the analysis of circulating miRNAs has been described as informative in the classification of patients with cardiovascular diseases^25,34, coronary artery disease²³, and chronic kidney disease. Furthermore, analysis of circulating miRNAs as biomarkers has shown great potential in the analysis and stratification of patients with different type of liver disease, including non alcoholic fatty liver disease [NAFLD³⁶] alcoholic liver disease [ALD³⁷] as well as and viral hepatitis type B and C^36-38.

Conclusion

One of the major challenges towards an improved treatment of human diseases is the identification of appropriate markers for an early detection of diseases as well as for monitoring disease progression and patients’ response to therapy. In this review, we discussed and highlighted the potential clinical utility of circulating miRNAs as blood biomarkers. Clearly the profiling of circulating miRNAs is still in its infancy; additionally, the development of dedicated methodological approaches has just begun. However, the wealth of information that has been already published strongly suggests that circulating miRNAs will maintain their promise as biomarkers and that this fields it is here to stay and to thrive. Furthermore, it easy to predict that not only the profiling of circulating miRNAs will find its way ‘away’ from the bench into the clinical and diagnostic environments, but that it will also find novel application such as monitoring the efficacy of anticancer or anti-viral therapies, as well as having the potential of becoming themselves (i.e. the circulating miRNAs) a future therapeutic targets for the treatment of human diseases.

About the Author

Mirco Castoldi is a molecular and cellular biologist that during his studies has explored different fields of basic research. Since achieving his PhD in 2004, he has joined the field of Molecular Medicine, where he investigated microRNA activity in the biogenesis of human disease. His fundamental contribution has lead to the development of microRNA expression profiling platform by using microarray (miCHIP) and qPCR (miQPCR). Recently, Mirco has discovered that the liver specific microRNA miR-122 has the potential to regulate systemic iron homeostasis. His current research focuses on elucidating the role played by microRNAs in controlling systemic and cellular iron homeostasis in health and disease.

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