MicroRNAs (miRNA) are single-stranded RNA molecules of about 21–23 nucleotides in length, which function in the regulation of gene expression. miRNAs are expressed as part of primary transcripts in the form of hairpins with signals for dsRNA-specific nuclease cleavage by the ribonuclease Drosha in combination with an RNA-binding protein. After the precursor miRNA is released as an approximately 70 nt RNA, it is transported from the nucleus to the cytoplasm by Exportin-5, and then is cleaved by Dicer RNase III to form a double-stranded RNA. Dicer initiates the formation of the RNA-induced silencing complex (RISC), which is responsible for the gene silencing observed due to miRNA expression and RNA interference [1], [2], [3].

MicroRNAs have been found in tissues and also in serum and plasma, and other body fluids, in a stable form that is protected from endogenous RNase activity (in association with RISC, either free in blood or in exosomes (endosome-derived organelles)). Studies by Lu et al [4] demonstrated the feasibility and utility of monitoring the expression of miRNAs in human cancer tissue. They found a high level of diversity in miRNA expression across cancers, and found that approximately 200 miRNAs could be sufficient to classify human cancers. Tam [5] adds that because miRNAs function as managers in gene regulatory networks, they are distinct from other biomarkers because they have a pathogenic role in the disease process and are not by-products of the disease state. Because miRNAs function by specific binding to their targets, polymorphisms (SNP) within the sequence of miRNAs or their target mRNAs can lead to disease, including cancer. These miRNA-specific SNPs can influence the risk of disease and can also be used in the diagnosis of these diseases [6]. Deregulated miRNAs have been described from numerous human cancers including breast, lung, colon, ovarian, and prostate cancer. Mitchell et al [7] found that miRNAs originating from prostate cancer tissue enter circulation and can be used to distinguish patients with prostate cancer from healthy controls and established a blood-based PCR approach for the detection of human prostate cancer. A similar approach was used to detect serum miRNA from ovarian cancer patients [8]. Taylor and Gercel-Taylor [9] investigated the use of circulating exosomes in the diagnosis of cancer. They found that the miRNA content of ovarian tumor cells and circulating exosome was similar and could be used to distinguish cancer patients from patients with benign ovarian disease and from normal controls.

miRNA signatures from normal and cancerous tissues have been used to classify several types of cancer and may also allow clinicians to determine a treatment course based on the original tissue type. It may also be possible to use miRNA expression patterns as a biomarker to monitor the effect of therapy on cancer progression [2]. Because miRNA expression profiles parallel the developmental origins of tissues, and because relatively few miRNAs can be used to effectively type tissues, they are potentially superior markers than messenger RNAs for cancer diagnosis [4]. The potential for the use of serum miRNAs as biomarkers of disease and as targets of therapeutics is promising [10], since it would mean a non-invasive, accurate test for cancer. In this study, we demonstrate that serum miRNAs can be used to discriminate between cancer patient sera and normal donor sera, using a simple microarray assay that requires no amplification step.

miRNA expression signatures have a potential role in the diagnosis, prognosis and therapy of human diseases, including cancer, heart disease, viral infections and inflammatory diseases [1]–[4], [10], [15]–[25]. Deregulation of miRNAs in cancer can be caused by chromosomal deletions, amplifications and translocations; by hypermethylation of CpG islands; and by regulation of transcription and post-transcriptional processing [1], [2], [23]. Aberrant expression of miRNAs can influence cancer progression by affecting the expression of oncogenes or tumor suppressors, and miRNAs, such as the miR-17-92 cluster, can function directly as oncogenes [1], [2], [23]. miRNAs are also involved in cancer through their effect on the cell cycle, apoptosis, metastasis, and angiogenesis [1], [2], [23].

Several studies have detailed the miRNAs that are associated with cancers [15], [23], [26]–[32]. Most of these studies have used biopsy samples, archival tissues or cancer cells [5], [33]–[37]; or have used miRNAs extracted from paraffin embedded or formalin fixed tissues [38]. By comparing non-cancerous tissues surrounding cancerous tissues, or normal donors versus cancer patients, those miRNAs that are up or down regulated can be identified, often after PCR amplification. Many studies have been published describing the specificity of miRNAs for different types of cancers, cancer stages and cancer treatments and several reviews have been published that summarize the most recent information on the roles of miRNAs in cancer [1], [2], [5], [6], [15], [23], [25], [32]. These studies demonstrate the utility of miRNAs in both diagnosis and prognosis of several cancers and also differentiate between cancer and benign disorders [34]. While numerous studies have led to an understanding of miRNAs at a tissue or cell level, there is a paucity of data in serum studies hampering their use in routine diagnosis.

Recently a series of studies has been performed on miRNAs present in serum [7]–[9], [39]–[41]. In a recent report, miRNAs were shown to be significantly elevated in pregnant versus non- pregnant women [40]; and in another study, placental miRNAs were shown to be present in maternal plasma [42]. In these studies, the miRNAs were found to be very stable to storage, and also to any RNAse degradation. miRNAs have recently been shown by Mitchell et al [7] to circulate in serum of prostate cancer patients [7]. In particular, miR-141 could differentiate prostate cancer patients from normal individuals [7]. In a work by Taylor and Gercel-Taylor [9], circulating tumor exosomes were isolated from serum of ovarian cancer patients using magnetic beads and an antiEpCAM antibody. miRNAs were then extracted, labeled and detected by microarray. This approach, using larger volumes of serum, indicated that eight diagnostic miRNAs were up-regulated in cancer exosomes: miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR-203, miR-205, and miR-214. To date however, no routine assay is available for examining miRNA signatures in serum or in the plasma of cancer patients.

One issue that appears in these studies is the fact that the miRNA expression patterns seen in serum are not identical to those seen from miRNAs taken directly from cancer cell lines. In our study, we found that although both the prostate cancer serum sample and the prostate cancer cell line (22Rv1) sample showed up-regulation compared to normal serum sample, they did not show much similarity to each other. This seeming discrepancy could be taking place for a number of reasons. The most obvious of which is the possibility that samples taken from the cell lines themselves are not representative of what appears in the serum. We can speculate that the most obvious source of miRNAs that appear in the serum is a product of tumor cell lysis; however, it may also be possible that their appearance in the serum is the product of a form of active transport involving the formation of exosomes (36). This would confound a direct comparison between miRNA expression patters derived from cell-line and tumor with those that are serum-derived.

Information on the use of miRNAs as biomarkers is predominantly associated with studies on tissue samples or cancer cell lines. Distinct patterns of miRNA expression are able to distinguish between cell type and stage in various cancers. This bodes well for diagnostic and prognostic applications of miRNA profiles. It also indicates there is clearly a need to define the expression profiles of miRNAs in serum of cancer patients and compare these to profiles observed in the serum of individuals representing a range of diseased and healthy states. It is anticipated that miRNA profiles in serum have the potential to be early markers for cancer detection and will also play a role in the monitoring of disease status during chemotherapy.

In this study, we have determined that a sufficient quantity of miRNAs is present in one ml of human serum to produce a detectable signal on a microarray using fluorescence or electrochemical detection. At the simplest level, this study has shown that serum miRNAs are up-regulated in cancer patients as compared to normal donors. In a comparison of stages 3 and 4 prostate cancer sera and normal donor serum miRNA levels, we found that 15 miRNAs (miR-16, -92a, -103, -107, -197, -34b, -328, -485-3p, -486-5p, -92b, -574-3p, -636, -640, -766, -885-5p) were up-regulated in serum from prostate cancer patients compared to normal donor sera.

Heat Map and cluster analyses show that serum miRNA signatures can also be used to separate cancer patients and normal donors in most cases. Sixty-five miRNAs (see Figure 6) were used in a Heat Map analysis that isolated 21 cancer samples, plus 3 re-assayed samples, from normal samples. Sixteen cancer samples clustered together, while five cancer samples formed a separate cluster within the normal sample cluster. Cluster analysis (see Figure 5) was also used to distinguish 21 cancer samples from 12 normal samples. Three cancer samples that were re-analyzed after freeze-thawing and storage, also clustered with the cancer samples

We have also shown that serum miRNAs can be detected at a level similar to that reported for TaqMan PCR from serum, approximately 4,000 copies per ul [7], and that the assay is stable for repeated sample testing after storage. The relative sensitivity of miRNA microarrays over standard expression arrays could be due to the hybridization kinetics of small oligonucleotides. Small molecules tend to have better hybridization kinetics than larger RNA or DNA molecules. Indeed, most of the thermodynamic models for predicting DNA hybridization behavior were developed using short oligonucleotides [43]. Many of the conclusions drawn from such studies do not apply when larger molecules are used as secondary structure, non-specific binding, and various other unforeseeable effects interfere with predicted hybridization behavior. For miRNAs, both their protection from digestion by various cellular factors, and their small size contribute to their detection in serum by microarrays at levels that are as low as those seen with methods that would otherwise be considered more sensitive, such as RT-PCR. The sensitivity of the microarray platform used here has allowed us to monitor miRNA from a small volume of serum and to classify sera as either from normal donors or from cancer patients.

Our results, in general, agree in many cases with previously published studies. However, differences in our results from other studies could result from several factors: 1) Serum miRNA expression profiles do not directly correspond to tissue profiles. The low levels of miRNAs in serum are better suited to studies of up-regulation and not down-regulation. In addition, it is unlikely that there is a direct correspondence between tissue miRNA levels and serum miRNA levels due to the possible mechanisms of miRNA release into circulation (cell lysis or exosome release [9]) [8], and to the release of miRNAs into circulation from other tissues as a result of the cancer or other related or non-cancer related conditions; 2) Different miRNA labeling techniques can produce different expression profiles. For example, ULS chemical labeling (Mirus and Kreatech kits), which only targets G residues, produces signal intensities that are directly proportional to the number of G residues in the miRNA. In contrast, labeling by incorporating modified nucleotides or label into an extended poly A tail, or by labeling with a modified primer by first strand cDNA synthesis, should produce a more even label; 3) We found that amplification techniques such as RT-PCR or T7 promoter expression, produce different expression patterns on arrays when compared to un-amplified samples. This could be due to differential efficiency in the amplification process. In addition, the limited quantity of miRNAs in human serum is not sufficient for column purification, which is commonly used for tissue miRNA purification.

An example of published data from two different miRNA expression profiling techniques that do not show strong agreement is illustrated in the following comparison. Schetter et al [44] labeled colon cancer tissue miRNAs by reverse transcriptase extension with a labeled primer and hybridized the target to a microarray, while Monzo et al [45] determined colon cancer tissue miRNA expression levels by TaqMan RT-PCR. When the 26 up-regulated miRNAs from the Schetter study are compared to those from the Monzo study, only 14 miRNAs are in agreement (54%). In a similar comparison of up-regulated prostate cancer tissue miRNAs from studies by Tong et al (TaqMan data [46]), Porkka et al (array data [47]), and Ambs et al (array and TaqMan data [33]), very little overlap of miRNA expression data can be seen: little or no overlap in data between Tong et al and Porkka et al, and only 1 of 33 and 1 of 34 miRNAs were in agreement for Tong et al compared to Ambs et al, and for Porkka et al compared to Ambs et al, respectively. Of the 15 upregulated serum miRNAs we report for prostate cancer, 4 are in agreement with data from Ambs et al, and 2 are in agreement with data from Porkka et al.

This study must be confirmed with a larger and better-documented data set. We do not yet know the affects of gender, age and cancer treatment on miRNA levels in serum. Radiation and chemotherapies that result in remission of cancer should also result in a change in the serum miRNA profiles. Wong, et al. [48] have shown that plasma levels of miR-184 were elevated in patients with squamous cell carcinoma of the tongue, and that plasma miRNA levels were reduced after surgical removal of the tumor. This would indicate that cancer treatment does have an affect on the levels of cancer-specific miRNAs in circulation. The samples used in this study were from cancer patients that were treated by chemotherapy in most cases. Because we do not have detailed information on the results of treatment on cancer progression or remission we cannot include this variable in our analysis. Additional studies with well-documented patient samples will be needed to address this question.