Extracellular microRNA: the promises for the diagnostics of multiple diseases

Small non-coding RNA molecules, microRNAs, are abundantly expressed in all cell types and are involved in the regulation of key cellular processes such as metabolism, proliferation, DNA repair, apoptosis and differentiation[1-3]. There have been more than 1500 different human miRNA species discovered so far and this amount is increasing[4]. Importantly, every miRNA has a unique nucleotide sequence and miRNA expression patterns are cell type specific[5,6]. Furthermore, deregulation of certain miRNAs expression in the cell was consistently observed during certain pathologies including cancer5. Several years ago, significant amounts of miRNA were detected in all biological fluids including blood plasma, urine, tears, breast milk, amniotic fluid, cerebrospinal fluid, saliva and semen[7]. These extracellular circulating miRNAs are surprisingly stable and survive unfavourable physiological conditions such as extreme variations in pH, boiling, multiple freeze thaw cycles and extended storage. In contrast to miRNAs, common RNA species like mRNA, rRNA and tRNA are degraded within several seconds after being placed in nuclease rich extracellular environment. Together with their unique sequences and unique tissue distribution, the high stability of extracellular miRNAs makes them ideal biomarkers from a clinical diagnostic point of view. The changes in miRNA spectra observed in certain biological fluids indeed correlate with various pathological conditions, further suggesting that extracellular miRNAs can serve as informative biomarkers to assess the pathological status of the body[8,9].

Several mechanisms explain the generation and existence of extracellular circulating miRNA. Primarily, miRNA can be passively released from dead or dying cells after plasma membrane rupture is triggered by mechanical injury, inflammation, apoptosis or necrosis[10]. Because intracellular miRNA binding proteins Argo – nautes are very stable in protease rich extracellular environments, they protect miRNA from degradation by nucleases[10,11]. Secondly, certain extracellular miRNAs are encapsulated into membrane microvesicles which are released by almost all cell types under normal and pathological conditions by either active or passive secretion mechanism[12,13]. Finally, some proportion of extracellular miRNA is floating in association with high-density lipoproteins (HDL) which, similarly to Argonaute proteins and microvesicles, are able to protect miRNA from degradation[14].

Both total miRNA concentration and its relative composition vary significantly among different fluid types[15]. While blood cells are significant contributors to extracellular miRNA in blood plasma and serum, miRNAs from other tissues are consistently detected in the circulation7. However, the extent to which certain organs contribute to the extracellular miRNA both in normal and pathological conditions remains to be studied in more detail. Importantly, upon toxicity in certain tissues, the levels of miRNAs that is specific for the affected tissues increase in the blood plasma. In a pioneering work, Laterza and co-authors documented significant increases in plasma concentrations of miR-122 (liver specific), miR-133a (muscle specific) and miR-124 (brain specific) in rats after injuries in liver, muscle and brain respectively[16]. Other researchers have shown that cardiac damage in human patients initiates the detectable release of cardiomyocyte-specific miRNA-208b and miRNA-499 into the circulation[17]. In patients with chronic hepatitis B, the increase of liver specific miR-122 in blood plasma strongly correlated with the severity of the disease[18]. More importantly, compared with an increase in aminotransferase activity (a common marker for liver damage) in the blood, the change in miR-122 concentration appeared earlier[18]. A tumour could also be considered an organ, and release miRNA into the bloodstream or other biological fluids. Indeed, many cancer tissue-specific miRNAs have been found in the blood circulation at different stages of the disease[19-22]. Furthermore, like all cell types, tumour cells secrete micro vesicles that contain their specific miRNA signatures[20-22]. These observations, along with many others, have established a basis for extracellular miRNA-based early diagnostics of human cancers.

Molecular methods used so far for the extracellular miRNA detection and characterisation were microarrays, next-generation sequencing (NGS) and qRT-PCR[8,9]. Microarrays had been widely exploited to define miRNA expression in early reports. However, due to their limited sensitivity, microarrays can only screen the most abundant miRNAs in biofluids. On the contrary, both qRT-PCR and NGS can detect low abundant miRNAs and thus were methods of choice in the majority of investigations. Despite the fact that NGS allows successful discovery of novel miRNAs, it is characterised by high-cost, labour-intensiveness and the requirement of a large amount of required RNA samples. Additionally, NGS data processing requires the analysis of the large number of miRNAs including ones whose changes are not associated with the disease and, therefore, not practically necessary. In contrast, qRT-PCR (including qPCR based arrays for the simultaneous detection of many miRNAs) is more convenient and cost-effective technique. Novel methods of more rapid, cost effective and accurate detection of circulating miRNA are likely to evolve in the future. For instance, Wang and colleagues have recently developed a nanopore sensor based on the α-haemolysin protein to selectively detect miRNAs at the single molecular level in blood plasma samples without the need for labels or amplification of the miRNA[23].

The sensor, which uses a programmable oligonucleotide probe to generate a target-specific signature signal, can quantify sub-picomolar levels of cancer-associated miRNAs and can distinguish single-nucleotide differences between different miRNA species[23]. The description of all reported miRNA signatures associated with certain diseased states goes well beyond the scope of this minireview. However, accumulated studies have so far compared the expression profiles of hundreds of extracellular miRNAs across a variety of non-malignant and malignant disorders to identify pathology-specific expression patterns[8,9]. It was consistently demonstrated that organ damage could be diagnosed by elevation of an organ specific miRNA expression in biological fluids[16-18]. Differences in viral and host miRNAs can be used to develop diagnostics tests of viral infections[24]. Expression profiles of miRNAs in transplant recipients can indicate their allograft status and predict the individual risk of rejection[25,26]. Because placental miRNAs can be detected in the maternal plasma, alterations in the miRNA profiles at various stages of pregnancy could serve as tools for pregnancy monitoring and prenatal diagnostics[27,28]. The diagnostic potential of extracellular miRNA for liver injury, cardiovascular, neurological, autoimmune, inflammatory and metabolic diseases has been also demonstrated and highlighted in a number of high quality reviews[8,9].

Cancers are often diagnosed in very late stages, which is what renders poor prognosis for the recovery and is mainly responsible for their stereotype of being ‘incurable diseases’. Efficient biomarkers to identify cancers at early stages, particularly during ordinary blood examination, do not exist at the moment. Despite the fact that numerous studies showed the amazing diagnostic potential of bio-fluids miRNA profiles for a variety of diseases, the overall focus was concentrated on oncological disorders including breast, prostate, ovarian, pancreatic, lung and colorectal cancers. Mitchell and coauthors were the first to demonstrate the presence of circulating tumour-derived miRNA in the blood plasma and serum of mice carrying prostate cancer xenografts[19]. They further showed that detection of prostate cancer expressed miR-141 in blood serum discriminate human patients with advanced prostate cancer from healthy controls[19]. In another study, Chen et al identified unique miRNA expression profiles in the blood serum of lung and colorectal cancer patients as compared to healthy subjects; specifically, the expression levels of miR-25 and miR-223 were significantly increased in non-small cell lung cancer sera[29]. Examining aberrant expression levels of miR-21, miR-486, miR-375 and miR-200b in sputum samples permitted distinguishing lung adenocarcinoma patients from healthy subjects with 80.6 per cent sensitivity and 91.7 percent specificity[30]. A set of miR-148b, miR-376c, miR-409-3p and miR-801 was shown to be significantly up-regulated in the plasma of breast cancer patients on early stages[31]. Another four plasma miRNA (miR-21, miR-210, miR-155 and miR-196a) were shown to diagnose pancreatic adenocarcinoma with a sensitivity of 64 percent and a specificity of 89 per cent[32]. Elevated plasma level of miR-25, miR-375 and let-7f clearly separated hepatocellular carcinoma cases from the healthy controls with specificity of 96 per cent and sensitivity of 100 per cent[33]. Besides these examples, there are hundreds of research publications describing abnormal miRNA expression profiles in a variety of biological fluids during the course of numerous cancers.

The utility of circulating tumour cells (CTCs) as prognostic markers in metastatic cancers has been well established; however, their efficacy and accuracy are still under question mainly due to the challenges associated with CTC enrichment and identification[34]. Recently, a panel of circulating miRNAs have been identified as biomarkers to predict the CTC status of metastatic breast cancer patients[35]. Thus, CTC-positive breast cancer patients had significantly higher levels of miR-141, miR-200a, miR-200b, miR-200c, miR- 203, miR- 210, miR-375 and miR-801 than CTC-negative patients and controls[35]. Therefore, the capacity of circulating miRNAs to indicate CTC status can help to overcome a fundamental limit of the sensitivity associated with current methods for CTC detection.

Despite obvious progress, the field of extracellular miRNA is still in its infancy phase and no tests based on miRNA biomarkers are available yet for clinical diagnosis. The one reason for this is relatively low reproducibility among the studies published on circulating miRNAs in cancers. The variations in the reported miRNAs can be partially explained by the fact that researchers are using different types of samples (plasma or serum). Furthermore, the lack of standardisation of the liquid sample processing protocols may render variability due to possibility of blood cell contamination in plasma / serum samples. Likewise, more studies are necessary to find accurate and stable endogenous control miRNAs to be used for the expression normalisation for each type of body fluid. Differences in study populations used (e.g. age, gender, cancer stages, ethnicity etc.) can also account for poor reproducibility between various reports. Finally, most if not all published investigations were concentrated on rather late stages of cancers and the capacity of circulating miRNA to detect tumours in either stage I or II has not been consistently reported. The primary reason for this is the limited availability of such patients since most cancers are predominantly diagnosed on much later stages. Secondly, optimisation of the protocols for the enrichment, isolation and detection of circulating miRNA is likely to be required to secure adequate sensitivity for detecting few copies of miRNAs.

Circulating miRNA biomarkers are seen as novel in non-invasive disease diagnostics, however, substantial work still needs to be done before they can be used in clinical settings. Before the discovery of extracellular miRNA, promise for early cancer diagnostics had laid within certain protein biomarkers (such as alpha-fetoprotein, carcinoembryonic antigen, carbohydrate antigen 15-3 and prostate-specific antigen) in blood plasma and other fluids. The clinical use of protein-based circulating tumour markers has been limited due to lack of sufficient sensitivity, specificity and stability. Indeed, methodological techniques of protein detection are fundamentally less sensitive than those of nucleic acids. Unlike proteins, nucleic acids can be detected by qPCR-based techniques via multimillion amplification of the signal and, thus, the presence of as few as one miRNA molecule in a given sample can be sufficient. Circulating miRNA profiles offer promises to detect tumours in early stages during ordinary blood tests in wide human populations. Additionally, extracellular miRNA signatures could identify the cancer patients who will have poor prognosis and need more aggressive treatment, while sparing the patients who will not.

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