Circulating microRNAs as stable blood-based markers for cancer detection

Abstract

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...

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

“…To this end, the abundance of the EV-associated miRNA let-7a and of the Argonaute 2-associated miR-16, reported as being excluded from EVs [45,55], was analysed.…”

“…Samples with a deviation >0.5 within the duplicates or with any evidence for melting curve abnormality were repeated. Spike-in normalisation with synthetic C. elegans derived cel-miR-54 sequence was performed to allow relative comparison across the analysed samples [45]. A normalised Ct value (Ctnorm) for let-7 or miR-16, was determined relatively to the syn-cel-miR-54 signal (Ctnorm = CtmiR-of-interest – Ctsyn-cel-miR-54).…”

The biology of personalized cancer medicine: Facing individual complexities underlying hallmark capabilities

“…The rapid implementation of miRNA profiling techniques (such as miRNA SAGE, microarray and deep‐sequencing) has helped in defining “miRNA gene signatures” for many cancer types, and several miRNAs now represent robust disease‐specific predictive and prognostic biomarkers (Kasinski and Slack, 2011). miRNA signatures can also be identified in the peripheral circulation of cancer patients (Mitchell et al., 2008), and several tumor‐associated miRNAs have been isolated from circulating microvesicles and exosomes (Rani et al., 2011). Of note, miRNAs are relatively stable compared to mRNAs and can be profiled from paraffin‐embedded tissues and body fluids such as blood and urine (Palmero et al., 2011; Wittmann and Jack, 2010).…”

Identification of aberrant circulating miRNAs in Parkinson's disease plasma samples

“…In the past years, miRNAs were found to be present in body fluids including blood, plasma, serum, saliva, urine, and milk (Mitchell et al., 2008). The extracellular miRNAs circulate in the blood of both healthy and diseased people, which are referred to circulating miRNAs.…”