Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (Ϸ22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumorderived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.biomarker ͉ miR-141 ͉ plasma ͉ serum ͉ prostate cancer T he development of minimally invasive tests for the detection and monitoring of common epithelial malignancies could greatly reduce the worldwide health burden of cancer (1). Although conventional strategies for blood-based biomarker discovery (e.g., using proteomic technologies) have shown promise, the development of clinically validated cancer detection markers remains an unmet challenge for many common human cancers (2). New approaches that can complement and improve on current strategies for cancer detection are urgently needed.MicroRNAs (miRNAs) are small (typically Ϸ22 nt in size) regulatory RNA molecules that function to modulate the activity of specific mRNA targets and play important roles in a wide range of physiologic and pathologic processes (3, 4). We hypothesized that miRNAs could be an ideal class of blood-based biomarkers for cancer detection because: (i) miRNA expression is frequently dysregulated in cancer (5, 6), (ii) expression patterns of miRNAs in human cancer appear to be tissue-specific (7), and (iii) miRNAs have unusually high stability in formalin-fixed tissues (8-10). This third point led us to speculate that miRNAs may have exceptional stability in plasma and serum as well. We show here that miRNAs are in fact present in clinical samples of plasma and serum in a remarkably stable form. Furthermore, we establish proof-ofprinciple for blood-based miRNA cancer detection by using both a xenograft model system and clinical serum specimens from patients with prostate cancer. Our results lay the foundation for the development of miRNAs as a novel class of blood-based cancer biomarkers and raise provocative questions regarding the mechanism of stability and potential biological function of circulating miRNAs. Results Identification and Molecular Cloning of Endogenous miRNAs fromHuman Plasma. Prior reports have suggested that RNA from human plasma (the noncellular component of blood remaining after removing cells by centrifugation) is largely of low molecular weight (11). W...

MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non-vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2-miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.icroRNAs (miRNAs) are a class of approximately 22 nucleotide noncoding RNAs that mediate posttranscriptional gene regulation by binding to and repressing specific messenger RNA targets. We and others previously demonstrated that miRNAs are present in the human circulation in a cell-free form and that altered plasma and serum miRNA profiles are observed in cancer and other diseases (1-9). This, along with the finding that miRNAs are remarkably stable in plasma despite high circulating RNase activity (1), suggests that miRNAs may be developed into a powerful new class of blood-based biomarkers.The mechanism underlying the unexpected stability of cell-free miRNAs in the RNase-rich environment of blood has not been systematically investigated, although it has important implications for miRNA biomarker development and for potential biological functions of circulating miRNAs (10). Currently, the dominant model for circulating miRNA stability is that miRNAs are released from cells in membrane-bound vesicles, which protect them from blood RNase activity. Vesicles proposed as carriers of circulating miRNAs include exosomes, which are 50-to 90-nm vesicles arising from multivesicular bodie...

MicroRNAs (miRNAs) are small (~22 nt) RNAs that play important roles in gene regulatory networks by binding to and repressing the activity of specific target mRNAs. Recent studies have indicated that miRNAs circulate in a stable, cell-free form in the bloodstream and that the abundance of specific miRNAs in plasma or serum can serve as biomarkers of cancer and other diseases. Measurement of circulating microRNAs as biomarkers is associated with some special challenges, including those related to pre-analytic variation and data normalization. We describe here our procedure for qRT-PCR analysis of circulating miRNAs as biomarkers, and discuss relevant issues of sample preparation, experimental design and data analysis.

Circulating, cell-free microRNAs (miRNAs) hold great promise as a new class of cancer biomarkers due to their surprisingly high stability in plasma, association with disease states, and ease of sensitive measurement. Yet little is known about the origin of circulating miRNAs in either healthy or sick people, or what factors influence levels of circulating miRNA biomarkers. Of 79 solid tumor circulating miRNA biomarkers reported in the literature, we found that fifty-eight percent (47/79) are highly expressed in one or more blood cell type. Plasma levels of miRNA biomarkers expressed by myeloid (e.g., miR-223, miR-197, miR-574-3p, let-7a) and lymphoid (e.g., miR-150) blood cells tightly correlated with corresponding white blood cell counts. Plasma miRNA biomarkers expressed by red blood cells (e.g., miR-486-5p, miR-451, miR-92a, miR-16) could not be correlated to red cell counts due to limited variation in hematocrit in the cohort studied, but were significantly increased in hemolyzed specimens (20-30 fold plasma increase; p<0.0000001). Finally, in a patient undergoing autologous hematopoietic cell transplantation, plasma levels of myeloid- and lymphoid-expressed miRNAs (miR-223 and miR-150, respectively) tracked closely with changes in corresponding blood counts. We present evidence that blood cells are a major contributor to circulating miRNA, and that perturbations in blood cell counts and hemolysis can alter plasma miRNA biomarker levels by up to 50-fold. Given that a majority of reported circulating miRNA cancer biomarkers are highly expressed in blood cells, we suggest caution in interpretation of such results as they may reflect a blood cell-based phenomenon rather than a cancer-specific origin.