MicroRNAs (miRNAs) are a class of non-coding small RNAs (18–24 nucleotides), which are endogenously stable and evolutionarily conserved molecules. The major role of miRNAs appears to be in acting as crucial switches in regulating posttranscriptional gene expression. In recent years, miRNAs have been identified to play an important role in many physiological and pathological conditions [1,2,3,4]. Particularly, miRNA deregulation in various cancer types has been extensively studied. For example, deregulations in miRNAs expression have been identified to play a role not only in major cancers like lung [5], breast [6] and prostate [7] but also in rare cancers like waldenstrom macroglobulinemia [8] and cholangiocarcinoma [9]. In this review, we summarize the research advancements of using miRNAs as circulating biomarkers in cancers. The prospect of miRNAs as potential diagnostic and prognostic biomarkers in a clinical setting is significant as more evidence points towards a central role of miRNAs in cancer development and progression.

microRNAs play a fundamental but significant role in cellular functions [10,11,12,13,14,15]. miRNA biogenesis and the mechanisms underlying miRNA mediated gene regulation are described elsewhere [16,17,18]. Since the discovery of miRNAs in C. elegans, about 1,500 miRNAs have been reported in humans [19]. Earlier studies established the function of miRNAs such as lin-4 and let-7 during embryogenesis and developmental control in C. elegans when complex gene regulations occur. Biochemical characterizations showed that these small RNAs block protein synthesis and/or regulate mRNA stability [20,21]. miRNAs have been found to regulate the expression of about 50% of the human coding gene transcripts [20]. The gene regulatory role of miRNAs suggests their important role in physiological functions [21] and pathological conditions [2,3]. Furthermore, single nucleotide polymorphisms in the 3’UTR of mRNAs may affect miRNA-mediated regulation and have been associated with certain cancer risk [22].

Pioneering studies by Calin et al. showed the biological relevance of miRNAs in cancer development; miR-15 and miR-16 were significantly downregulated in chronic lymphoid leukemia (CLL) through deletions in chromosome 13q14 locus. These earlier observations also revealed 52% of miRNA genes to be located in genomic regions, which are frequently altered in cancer. These results were further validated by mapping which revealed 186 miRNAs located at fragile sites and genomic regions [23]. Since this discovery, miRNAs have been implicated in many different types of cancers including the influence of miRNAs in neoplastic transformation, progression and patient outcomes [24,25,26,27,28,29]. miRNAs act as oncogenes or tumor suppressors taking the lead role in regulating cancer pathways. The target genes regulated by the miRNAs determine their role as oncogenes or tumor suppressors.

With our increasing knowledge on gene regulatory pathways, we have achieved certain milestones in our understanding of cancer signaling. However, there is an immense need for fail proof biomarkers that can detect cancer in its earlier stages and/or function as prognostic indicators. Currently, most cancer diagnosis depends on imaging techniques like computerized tomogram (CT), magnetic resonance imaging (MRI), ultrasonography, positron emission tomography (PET), mammogram and invasive studies like colonoscopy, bronchoscopy and biopsy. Even though imaging and invasive studies are extensively relied on in order to generate valid information regarding the cancer, there is a high possibility of false positive or false negative results. The imaging methods are not cost effective and invasive studies cause unnecessary discomfort for the patients.

Further, the available biomarkers such as CA 125 for ovarian cancer, CA 19-9 for pancreatic cancer and CEA (carcino embryonic antigen) for colon cancer have poor sensitivity and specificity. Only 50% of patients with early stage ovarian cancer have elevated levels of CA125. In 2010, the National Institutes of Health advisory panel did not reissue the old protocol which advised women to begin yearly breast cancer screening once they attained the age of forty. Furthermore, the U.S. Preventive Services Task Force (USPSTF) recommendation on prostate cancer screenings (PSA) made available in October 2011 recommends discontinuing routine PSA tests due to poor sensitivity and specificity in distinguishing prostate cancer patients requiring surgery. Thus, there is a critical need for reliable biomarkers that allow precise monitoring of changes at the cellular level and the patient’s response to therapy. miRNAs have tremendous potential to be explored as diagnostic and prognostic biomarkers. miRNA expression is dynamic; many miRNAs are deregulated in early stages of tumor development and are upregulated during cancer progression exhibiting their potential for diagnostic utility [36,37]. The possibility of using combination of markers such as CA-19-9 and miRNAs significantly increase the diagnostic potential [38].

Resnick et al. compared levels of miRNAs (miR-21, -92 and -93) and the currently available biomarker for ovarian cancer, CA-125, and showed alteration in the level of these miRNAs in patients with ovarian cancer having normal levels of CA-125 [39]. With increasing implications of miRNAs in cancer development and progression, significant efforts are underway to use miRNAs as novel biomarkers with clinical applications [40] and as novel therapeutic targets for anti cancer drug resistance [41]. Another potential is in the diagnosis of pancreatic cancer, which still remains a cancer with poor prognosis with a median survival of 6 months to 1 year. Pancreatic cancer is asymptomatic in the early stages when their precursor lesions such as PanIN (pancreatic intraepithelial neoplasia) remain undetected. However, if these precursor lesions are detected, the potential for cure may be high. Several miRNAs have been identified in PanIN stages [42,43] of pancreatic cancer that allow us to investigate them as potential biomarkers.

Blood sampling from patients remain the least invasive method for identifying biomarkers. The presence of circulating miRNAs has been demonstrated in various disease conditions and the profiles vary with the degree of disease progression [44,45,46,47,48]. The presence of a specific set of circulating miRNAs in cancer condition is justified by passive diffusion due to high turnover of cells in cancer. Yu et al. showed differences between the metabolite profiles of plasma and serum. Concentrations of metabolite were generally higher in serum and provided higher sensitivity than plasma [49]. Initial studies to identify serum miRNAs in cancer were demonstrated by Lawrie et al., in which they observed that miR-155, miR-210, and miR-21 were upregulated in diffuse large B cell lymphoma when compared to healthy controls [50]. Further, miRNA expression profiles have been generated using serum/plasma from patients diagnosed with common cancers such as acute leukemia [51], breast cancer [52], colorectal cancer [53], gastric cancer [54], glioblastoma [55], hepatocellular cancer [56], lung cancer [57], oral and squamous cancer [58], ovarian [39] and prostate cancer [40]. Recently, miRNAs have also been detected in numerous other body fluids (see below).

With the increasing data about serum miRNAs as well the growing need for a good biomarker, which must also be easily accessible, the miRNAs in serum can be considered valid biomarkers for early detection, diagnosis and prognosis of various cancers [59,60,61,62,63,64]. As more interest has been drawn toward identifying circulating miRNAs in cancers, the biggest challenge currently encountered during the analysis of miRNAs in serum/plasma and other body fluids is the need for an internal normalization. Housekeeping genes like U6 or GAPDH, commonly used as internal normalization during miRNA analysis in cells and tissues, are not feasible in serum analysis since these controls are easily degraded and not detected in serum. Chen et al. [57] demonstrated a series of miRNAs existing in normal human serum and compared their levels to widely used U6, which was not stable in serum. They also showed the presence of miRNAs in the serum of rats, mice, calves, bovine fetuses and horse and the potential for using these miRNAs for normalization and comparative studies. In support of these observations, Mitchell et al. demonstrated the stability of miRNAs (miR-15b, miR-16, miR-19b and miR-24) occurring in normal human sera and their resistance to RNase digestion. The levels of these miRNAs are not altered during tumor growth and can possibly be considered as normalization controls [40]. Of these normally present miRNAs, miR-16 was widely present and was used as internal controls in serum miRNA studies [57]. However a recent study demonstrated high levels of miR-16 in red blood cells; thus it is possible that the presence of miR-16 in serum may in part be due to hemolysis of red blood cells [65]. This also raises the question of using miR-16 for normalization. As greater numbers of miRNAs are being detected in normal blood cells, to avoid the errors with internal control there is an essential need for a standard, normalization protocol when analyzing miRNAs from serum samples. This challenge is, for now, met by using spike-in synthetic C. elegans miRNA for internal normalization when using serum samples for miRNA analysis. Taken together, circulating miRNAs detected using validated methods can be valuable biomarkers for cancer diagnosis and prognosis. A comprehensive list of miRNAs identified as potential biomarkers in various cancer types are given in Table 1.

In an attempt to establish miRNAs as biomarkers in cancer, considerable interest has been drawn to the ability of detecting these circulating biomarkers in different body fluids and the stability of miRNAs to resist the numerous extracellular enzymes present in human body. Weber et al., showed the presence of miRNAs in different body fluids such as plasma, saliva, tears, urine, amniotic fluid, colostrum, breast milk, bronchial lavage, cerebrospinal fluid, peritoneal fluid, pleural fluid, seminal fluid and serum [100]. There is also evidence of miRNAs being identified in different body fluids in various cancer types, some of them being serum miRNAs in prostate cancer [101], urine miRNAs in urothelial carcinoma [102], cerebrospinal fluid (CSF) miRNAs in glioma [103], plasma miRNAs in pre-eclampsia [104] and sputum miRNAs in non small cell lung cancer (NSCLC) [105]. These studies provide strong evidence to indicate the existence of miRNAs in different body fluids. The stability of endogenous miRNAs to RNase digestion was shown by adding synthetic miRNAs to the serum from subjects with prostate cancer. The added synthetic miRNAs were destroyed whereas the endogenous miRNAs were able to maintain their stability and were detected in plasma/serum [40]. In yet another study, Chen et al. elucidated the stability of miRNAs in various body fluids, after the miRNAs were subjected to a series of experimental conditions such as boiling, low/high pH, extended storage and multiple freeze thaw cycles [57].

Recent findings have demonstrated the presence of miRNAs in exosomes, microvesicles, lipoproteins, apoptotic bodies and large micro particles. Valadi et al. analyzed human and mouse mast cell lines and observed the synthesis of new proteins on transfer of exosomal RNA between the donor cells and the recipient cells. They proposed that the exosomes contained both mRNA and miRNA and a possible mechanism of genetic exchange between the cells [106]. It is probable that the stability of endogenous miRNAs is imparted by the protection of miRNAs in membrane bound exosome-like particles called microvesicles [107]. Gallo et al. recently observed that a majority of miRNAs in saliva and serum from healthy individuals and systemic lupus erythmatosus (SLE) patients were found to be concentrated in exosomes [108].

Evidence from recent studies has also proven the presence of miRNAs in high-density lipoproteins (HDL) [109]. The study by Vickers et al. showed that HDL has a major role in endogenous miRNA transport. They isolated HDL from normal human plasma and from patients with familial hypercholesterolemia by density gradient ultracentrifugation and fast-protein liquid chromatography followed by anti-apoA-1 immunoprecipitation. Total RNA was extracted from purified HDL and miRNA profiles analyzed using microarrays. They demonstrated occurrence of various miRNAs in HDL molecules, and one of the abundant miRNA found in both human and mouse HDL was miR-223 [110]. Furthermore, they evaluated dynamic response by subjecting various cell lines to stress. The results revealed a number of proteins involved in the exportation process and suggested nucleophosmin 1 (NPM1) as a protective protein guarding miRNA from degradation. These findings about the stability of endogenous miRNA and protection from degradation outside the cellular environment imparted by exosomes and protective proteins suggest possible reasons for their detection in plasma/serum.

miRNAs are revolutionizing the field of cancer research. These small RNAs are able to distinguish cancer types and subtypes and have the potential to be biomarkers in cancers. However, there is a need to address the level of correlation that exists between the miRNA identified in tumor tissue and circulating miRNAs. Further studies establishing miRNA expression patterns in relation to patient’s age, demography, common health conditions and lifestyles are needed. Before developing serum miRNA as a clinical biomarker, the techniques of miRNA extraction, quantification, data analysis and a normalization strategy needs to be standardized. Currently, one of the main challenges in analyzing miRNA from serum samples is the normalization factor. The effective use of animal models will significantly increase the success of developing suitable miRNA biomarkers in cases where the access to human patient samples is limited [118]. Effectively addressing these challenges will help us overcome some of the current pitfalls and establish miRNAs as fail proof biomarkers in cancer diagnosis and treatment.